Genetically engineered clostridial genes, proteins encoded by the engineered genes, and uses thereof

ABSTRACT

The present invention relates to an isolated Clostridial neurotoxin propeptide having a light chain region, a heavy chain region, where the light and heavy chain regions are linked by a disulfide bond, and an intermediate region connecting the light and heavy chain regions. An isolated nucleic acid molecule encoding a Clostridial neurotoxin propeptide is also disclosed. Also disclosed is an isolated, physiologically active Clostridial neurotoxin produced by cleaving a Clostridial neurotoxin propeptide, a vaccine or antidote thereof, and methods of immunizing against or treating for toxic effects of Clostridial neurotoxins. Methods of expressing recombinant physiologically active Clostridial neurotoxins are also disclosed. Also disclosed is a chimeric protein having a heavy chain region of a Clostridial neurotoxin and a protein with therapeutic functionality. A treatment method is also disclosed.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 60/630,175, filed Nov. 22, 2004, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to isolated Clostridial propeptides andneurotoxins, vaccines or antidotes thereof, methods of immunizing andtreating subjects, isolated nucleic acid molecules encoding Clostridialpropeptides and neurotoxins, methods of expression, chimeric proteins,and treatment methods.

BACKGROUND OF THE INVENTION

The Clostridial neurotoxins are a family of structurally similarproteins that target the neuronal machinery for synaptic vesicleexocytosis. Produced by anaerobic bacteria of the Clostridium genus,botulinum neurotoxins (“BoNT”s, seven immunologically distinct subtypes,A-G) and Tetanus neurotoxin (“TeNT”) are the most poisonous substancesknown on a per-weight basis, with an LD₅₀ in the range of 0.5-2.5 ng/kgwhen administered by intravenous or intramuscular routes (NationalInstitute of Occupational Safety and Health, “Registry of Toxic Effectsof Chemical Substances (R-TECS),” Cincinnati, Ohio: National Instituteof Occupational Safety and Health (1996)). BoNTs target cholinergicnerves at their neuromuscular junction, inhibiting acetylcholine releaseand causing peripheral neuromuscular blockade (Simpson, “Identificationof the Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol.Toxicol. 44:167-193 (2004)). BoNT serotypes A, B, and E are consideredto represent the most significant threat to military and civilianpopulations, particularly because they can be aerosolized and deliveredby inhalation (Arnon et al., “Botulinum Toxin as a Biological Weapon:Medical and Public Health Management,” JAMA 285:1059-1070 (2001)).

Though much work has been done to develop vaccines or antidotes whichare effective against poisoning with Clostridial neurotoxins, theeffectiveness of available products is limited because the availableinactivated toxin preparations do not optimally mimic the native toxin.No therapeutic antidotes or vaccines have been approved for widespreaduse, though some preparations are available for limited use underspecific circumstances. The NIAID Biodefense Research Agenda hasidentified the development of countermeasures against Clostridialneurotoxins as one of its most pressing goals (National Institute ofAllergy and Infectious Diseases, “NIAID Biodefence Research Agenda forCDC category A Agents” NIH Publication #03-5308 (2002)). A prime targetis understanding and preventing neurotoxin entry into target cells.Immunological approaches have utilized passive protection via injectionof antibodies as antitoxins, or active immunization via vaccination withtoxoids, toxins chemically or genetically transformed to render themnon-toxic but still immunogenic (Ramon et al., “Sur L'immunizationAntitetanique et sur la Production de L'antitoxine Tetanique,” Compt.Rend. Soc. Biol. 93:508-598 (1925)). Antibody-based anti-toxins areavailable in limited quantities, but no protective vaccine againstClostridial neurotoxins has been approved. A pentavalent botulinumtoxoid (ABCDE), consisting of toxins inactivated by temperature orcross-linked with formaldehyde, is available in limited quantities, andhas been shown to induce antibodies in laboratory workers and militarypersonnel (National Institute of Allergy and Infectious Diseases, “NIAIDBiodefence Research Agenda for CDC category A Agents. Progress Report,”NIH Publication #03-5435 (2003)). An inactivated heavy chain toxoidadministered by inhalation was found to protect animals against inhaledtoxin doses 10⁴ times the LD₅₀ (Park et al., “Inhalational Poisoning byBotulinum Toxin and Inhalation Vaccination with Its Heavy-ChainComponent,” Infect. Immun. 71:1147-1154 (2003)). An investigationalheptavalent antitoxin (A-G reactive, equine origin) against BoNT isbeing developed by the U.S. Department of Defense and is now beingtested. Initial data demonstrate the general safety of this antitoxin,though it displays some cross-species reactogenicity in humans. Anotherinvestigational BoNT anti-toxin is based on a combination of threerecombinant monoclonal antibodies, which neutralize BoNT A with a highpotency (Nowakowski et al., “Potent Neutralization of BotulinumNeurotoxin by Recombinant Oligoclonal Antibody,” Proc. Natl. Acad. Sci.USA 99:11346-11350 (2002)). Development and testing of human monoclonalantibodies to BoNT B-G is also currently in progress and supported byNIAID (National Institute of Allergy and Infectious Diseases, “NIAIDBiodefence Research Agenda for CDC category A Agents. Progress Report,”NIH Publication #03-5435 (2003)).

Several laboratories are attempting to develop recombinant Clostridialtoxin genes or fragments thereof. The Department of Defense hasdeveloped a vaccine based on expression of the receptor-binding domainof the BoNT A heavy chain (National Institute of Allergy and InfectiousDiseases, “NIAID Biodefence Research Agenda for CDC Category A Agents.Progress Report,” NIH Publication #03-5435 (2003); Byrne et al.,“Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type ABinding Domain from Pichia pastoris as a Recombinant Vaccine Candidate,”Infect. Immun. 66:4817-4822 (1998); and Pless et al., “High-Affinity,Protective Antibodies to the Binding Domain of Botulinum Neurotoxin TypeA,” Infect. Immun. 69:570-574 (2001)). A similar approach with arecombinant BoNT F fragment expressed in Salmonella typhimurium wasfound to provide partial protection of animals against the toxin (Foyneset al., “Vaccination Against Type F Botulinum Toxin Using AttenuatedSalmonella enterica var Typhimurium Strains Expressing the BoNT/F H_(C)Fragment,” Vaccine 21:1052-1059 (2003)). A catalytically activenon-toxic derivative of BoNT A expressed in E. coli was reported toinduce toxin-neutralizing antibodies and protect animals from a BoNTchallenge (Chaddock et al., “Expression and Purification ofCatalytically Active, Non-Toxic Endopeptidase Derivatives of Clostridiumbotulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002)). Acatalytically inactive, full-length derivative of BoNT C expressed in E.coli was immunogenic in mice, though limitations of this system hinderexpression of full-length native and active recombinant toxin (Kiyatkinet al., “Induction of an Immune Response by Oral Administration ofRecombinant Botulinum Toxin,” Infect. Immun. 65:4586-4591 (1997)).Rummel et al. (“Synaptotagmins I and II Act as Nerve Cell Receptors forBotulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004) (“RummelI”)) and Rummel et al. (“The H_(cc)-domain of Botulinum Neurotoxins Aand B Exhibit a Singular Ganglioside Binding Site DisplayingSerotype-Specific Carbohydrate Interaction,” Mol. Microbiol. 51:631-643(2004) (“Rummel II”), report full-length BoNT A, B, and G neurotoxinsexpressed in an E. coli from plasmids encoding the respectivefull-length genes. Rummel I and Rummel II also report severalderivatives of BoNT genes. The neurotoxins described in Rummel I andRummel II are active only at very high concentrations. This is likelydue to the fact that the neurotoxins expressed by Rummel I and Rummel IIare denatured during expression, extraction, and purification from E.coli and achieve low physiological activity of the single chain BoNTpropeptide due to improper disulfide bonding. Thus, although Rummel Iand Rummel II may in fact have produced full-length recombinant BoNTpeptides of serotypes A, B, and G, the properties of the neurotoxinsdescribed do not possess native structures and physiological activity.

The widely used E. coli expression system may be problematic for someproteins, because the E. coli cytosol may not provide the non-reducingenvironment needed for maintenance of disulfide bridges critical to thenative toxin structure (Alberts et al., Molecular Biology of the Cell,Third Edition, Garland Publishing Inc., 112, 113, 488, 589). Inaddition, E. coli based expression systems also present practicalproblems associated with endotoxin removal. These limitations emphasizethe importance of selecting an expression system capable of producingrecombinant molecules that retain the native toxin structure andbiological activity.

Data from multiple laboratories suggest that the C-terminal moiety ofClostridial toxin heavy chains (“Hc”), or the intact heavy chain (“HC”)expressed or prepared by reduction/denaturation from native toxins, arefunctionally altered and therefore require a ˜10,000-fold molar excessto delay the onset of toxin-induced paralysis (Li et al., “RecombinantForms of Tetanus Toxin Engineered for Examining and Exploiting NeuronalTrafficking Pathways,” J. Biol. Chem. 276:31394-31401 (2001); Lalli etal., “Functional Characterization of Tetanus and Botulinum NeurotoxinsBinding Domains,” J. Cell Sci. 112:2715-2724 (1999)). Some of thesepreparations have been completely inactive in this assay(Daniels-Holgate et al., “Productive and Non-Productive Binding ofBotulinum Neurotoxin A to Motor Nerve Endings are Distinguished by ItsHeavy Chain,” J. Neurosci. Res. 44:263-271 (1996)). The low efficiencyof HC and Hc may be due to either their increased binding affinity tonon-productive sites on cells normally mediating toxin trafficking ortheir conformational differences from the native toxin which results ina low binding affinity for the specific binding sites at the targetcells. In either case, incorrect folding, altered post-translationalmodification, a requirement for the N-terminal portion of the molecule(Koriazova et al., “Translocation of Botulinum Neurotoxin Light ChainProtease through the Heavy Chain Channel,” Nat. Struct. Biol. 10:13-18(2003)), or multiple other changes, may be responsible for thesefunctionally important deficiencies. These facts suggest that thecurrently available preparations of BoNT or its derivatives are poormimics of the native toxin, which may limit their therapeutic potential.

The methods currently available to produce inactivated derivatives ofBoNTs as vaccines or antidotes to BoNT poisoning have met with limitedsuccess. This can be due to several factors. First, the methods used toinactivate BoNT prepared from Clostridial cultures are harsh, and mayalter the toxin's native conformation in ways that may influence itsimmunogenicity or trafficking and absorption. Second, methods based onproducing recombinant toxins have thus far only succeeded in producingeither inactive toxin molecules or fragments of its protein domains. Inboth cases, the recombinant molecules produced are by definitionsignificantly different from native toxin, particularly with respect topost-translational processing and disulfide bonding. Though inactivatedtoxins and toxin fragments have been shown to be immunogenic, the poolof polyclonal antibodies they generate will include a fractionrecognizing epitopes present only on misfolded toxins.

Another area in which Clostridial neurotoxins have been extensivelystudied relates to their clinical use to treat dystonias, and totemporarily correct aesthetic defects in skin. These indications arespecific to the neurotoxins produced by strains of Clostridium botulinum(BoTox), because they can be used at extremely small doses to locallyparalyze specific muscles and thereby achieve therapeutic goals. All ofthe current products used for this indication are produced fromClostridial cultures, and there have been no reports of an active BoToxmolecule produced using any type of genetic engineering technology.

A further area of interest is derived from the ability of Clostridialneurotoxins to pass undegraded through epithelial barriers viatranscytosis, and specifically target nervous tissue. This has led tosuggestions that Clostridial neurotoxins can be used to enable oral andinhalational carriers for therapeutic agents that cannot normally bedelivered via these routes of administration, and delivery vehicleswhich can specifically target the peripheral and central nervous system.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to an isolated Clostridialneurotoxin propeptide. The propeptide has a light chain region, a heavychain region, where the light and heavy chain regions are linked by adisulfide bond, and an intermediate region connecting the light andheavy chain regions. The intermediate region has a highly specificprotease cleavage site which has three or more specific adjacent aminoacid residues that are recognized by the highly specific protease inorder to enable cleavage.

Another aspect of the present invention relates to an isolated nucleicacid molecule encoding the above Clostridial neurotoxin propeptide aswell as expression systems and host cells containing this nucleic acidmolecule.

A further aspect of the present invention relates to an isolated,physiologically active Clostridial neurotoxin produced by cleaving theabove Clostridial neurotoxin propeptide. The propeptide is cleaved atthe highly specific protease cleavage site. The light and heavy chainregions are linked by a disulfide bond.

Yet another aspect of the present invention relates to a vaccine orantidote including the above physiologically active, atoxic, Clostridialneurotoxin produced by cleaving the isolated Clostridial neurotoxinpropeptide at the highly specific protease cleavage site. The light andheavy chain regions are linked by a disulfide bond.

Still another aspect of the present invention relates to method ofimmunizing a subject against toxic effects of a Clostridial neurotoxin.This method involves administering the above vaccine to the subjectunder conditions effective to immunize the subject against toxic effectsof Clostridial neurotoxin.

Yet a further aspect of the present invention relates to a method oftreating a subject for toxic effects of a Clostridial neurotoxin. Thismethod involves administering an antidote comprising the abovephysiologically active, atoxic, Clostridial neurotoxin produced bycleaving the isolated Clostridial neurotoxin propeptide under conditionseffective to treat the subject for toxic effects of Clostridialneurotoxin.

Still a further aspect of the present invention relates to a chimericprotein including a first protein or protein fragment having a heavychain region of a Clostridial neurotoxin and a second protein or proteinfragment linked to the first protein or protein fragment.

Another aspect of the present invention relates to a method ofexpressing a recombinant physiologically active Clostridial neurotoxin.This method involves providing a nucleic acid construct having a nucleicacid molecule encoding an isolated Clostridial neurotoxin propeptide.The nucleic acid construct has a heterologous promoter operably linkedto the nucleic acid molecule and a 3′ regulatory region operably linkedto the nucleic acid molecule. The nucleic acid construct is introducedinto a host cell under conditions effective to express thephysiologically active Clostridial neurotoxin.

A further aspect of the present invention relates to a treatment method.This method involves contacting a patient with an isolated,physiologically active, toxic, Clostridial neurotoxin produced bycleaving the above isolated Clostridial neurotoxin propeptide.

The present invention relates to a genetic engineering platform thatenables rationale design of therapeutic agents based on Clostridialtoxin genes. The genetic engineering scheme is based on a two-stepapproach. For each Clostridial toxin serotype, gene constructs,expression systems, and purification schemes are designed that producephysiologically active, recombinant Clostridial neurotoxin. This ensuresthat the recombinant toxin derivatives retain structural featuresimportant for developing therapeutic candidates, or useful biologicreagents. Using the genetic constructs and expression systems developedby this paradigm, selective point mutations are then introduced tocreate atoxic recombinant derivatives. This two-step approach isdesigned to ensure that the recombinant toxin derivatives retain theimmunogenicity, absorption profile, and trafficking pathways of nativetoxin, allowing the atoxic derivatives to have optimized therapeutic andbiological properties. They also enable useful chimeric proteins to becreated.

Genetically engineered forms of recombinant toxins which structurallyand functionally mimic native toxins are superior to the toxoidscurrently in development for therapeutic purposes. They provide newapproaches which can produce customized toxin derivatives in largequantities, and with mutations specifically targeted to the creation ofvaccines and toxin antidotes. By focusing on solving the problemsassociated with producing recombinant toxins, which are physiologicallyactive, the inactivated toxin derivatives of the present invention havedistinct advantages over currently available alternatives. This isparticularly true with respect to their immunogenic activity and theirability to compete with native toxin for cellular binding sites.

The methodology described herein has additional scientific and practicalvalue because it provides a broad platform enabling facile manipulationand expression of Clostridial toxin genes. This will facilitate studiesof the mechanism of Clostridial toxin action, their intracellulartrafficking, and the factors responsible for their ability to transitthrough specific cell types without activation or toxic consequences. Inaddition, the BoNT constructs created can provide new tools fordelivering specific reagents or drugs via oral or inhalation routes, orspecifically into peripheral neurons, and enable their controlledactivation at the site of intended action. Other approaches to engineerdelivery tools based on chemically modified heavy chains fromClostridial neurotoxins have had limited success, possibly because themethods used to inactivate the toxin interfere with protein spatialstructure (Goodnough et al., “Development of a Delivery Vehicle forIntracellular Transport of botulinum Neurotoxin Antagonists,” FEBS Lett.513:163-168 (2002), which is hereby incorporated by reference in itsentirety).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show comparative alignment of amino acid sequences of theseven wildtype botulinum neurotoxin serotypes, including Clostridiumbotulinum serotype A (SEQ ID NO: 1), Clostridium botulinum serotype B(SEQ ID NO: 2), Clostridium botulinum serotype C (SEQ ID NO: 3),Clostridium botulinum serotype D (SEQ ID NO: 4), Clostridium botulinumserotype E (SEQ ID NO: 5), Clostridium botulinum serotype F (SEQ ID NO:6), and Clostridium botulinum serotype G (SEQ ID NO: 7). Gaps have beenintroduced to maximize homology. Amino acids identical in ≧50% ofcompared sequences are shown in black boxes. Amino acids constitutingthe active site of the catalytic domain of metalloprotease are marked bystars. Disulfide bridge between neurotoxin cysteine residues of thelight and heavy chain are shown as a long horizontal bracket. The aminoacid residues constituting the minimal catalytic domain of the lightchain are hatched. The first amino acid of the C-terminal part of theprotein heavy chain (N872 for BoNT A), constituting receptor-bindingdomain are shown with the arrow. Amino acids, absent in the maturedichain BoNT A molecule along with the aligned amino acids of the otherBoNT serotypes are boxed. The white arrow is positioned at the firstamino acid of the neurotoxins' heavy chain.

FIGS. 2A-C show comparative alignment, using the Clustal Program, ofamino acid sequences of the seven botulinum neurotoxin serotypes,including Clostridium botulinum serotype A (SEQ ID NO: 8), Clostridiumbotulinum serotype B (SEQ ID NO: 9), Clostridium botulinum serotype C(SEQ ID NO: 10), Clostridium botulinum serotype D (SEQ ID NO: 11),Clostridium botulinum serotype E (SEQ ID NO: 12), Clostridium botulinumserotype F (SEQ ID NO: 13), and Clostridium botulinum serotype G (SEQ IDNO: 14), which have been slightly modified in accordance with thepresent invention. Gaps have been introduced to maximize homology. Aminoacids identical in ≧50% of compared sequences are shown in black boxes.Amino acids constituting the active site of the catalytic domain ofmetalloprotease are marked by stars. Disulfide bridge between neurotoxincysteine residues of the light and heavy chain are shown as a longhorizontal bracket. The amino acid residues constituting the minimalcatalytic domain of the light chain are hatched. The first amino acid ofthe C-terminal part of the protein heavy chain (N876 for BoNT A),constituting receptor-binding domain are shown with the arrow. Aminoacids, absent in the mature dichain BoNT A molecule along with thealigned amino acids of the other BoNT serotypes are boxed. The whitearrow is positioned at the first amino acid of the neurotoxins' heavychain. Amino acid residues are modified in comparison with the wild typesequence to restrict trypsin-like proteolysis. Amino acids whichconstitute the insertion/modification into the wild type amino acidresidues and represent an enterokinase cleavage site are also shown.

FIGS. 3A-B illustrate features of the wild type BoNT A protein and gene(wt), and its toxic recombinant derivative (td). FIG. 3A is a schematicrepresentation of the native BoNT A (wt) dimer, illustrating thecatalytic (˜50 kDa), translocation (˜50 kDa), and receptor-binding (˜50kDa) domains. FIG. 3B is a comparison of the nucleotide (SEQ ID NO: 65(wt) and SEQ ID NO: 66 (td)) and amino acid (SEQ ID NO: 1 (wt) and SEQID NO: 8 (td)) sequences of the native BoNT A (wt) and its recombinanttoxic derivative (td), as generated in plasmid pLitBoNTA. Sequencescommon to both the wt and td genes are shown as black letters on a whitebackground, or as white boxes. White letters on a black backgroundrepresent the amino acids excised from the toxin propeptide to generatethe mature wt toxin. The disulfide bonds joining the LC and HC are shownas long horizontal brackets. Grey letters indicate the uniqueendonuclease restriction sites introduced into non-coding portions ofthe td DNA sequence and the Shine-Dalgarno region of the wt sequence.All other mutations introduced to modify the construct properties arealso shown in grey letters. The de novo enterokinase cleavage siteinserted into the td propeptide is shown by an arrow. Amino acidsproximal to conceived (wt) or executed (td) mutations are numbered.

FIGS. 4A-B show expression and purification of the toxic derivative ofBoNT A (td) in E. coli. FIG. 4A shows 8% PAGE stained with CoomassieG-250. FIG. 4B shows a Western blot of the PAG shown in FIG. 4A, probedwith polyclonal antibodies raised against the full-length BoNT A toxoid.Samples were treated with β-mercaptoethanol before separation. Theprotein molecular weight standards are shown to the far left. Lanes 1and 2 are cleared lysate of E. coli transformed with pETcoco2 emptyvector (Lane 1) or pETcocoBoNTA (Lane 2). Lane 3 is a purifiedpreparation of native BoNT A used as positive control. Lane 4 and 5 areeluates from the Ni-NTA affinity purification of cleared E. coli lysateswhich have been transformed with pETcoco2 (Lane 4) or pETcocoBoNTA (Lane5). SC: single chain propeptide. HC: Heavy Chain. LC: Light Chain.

FIG. 5 is a schematic representation of the three recombinant BoNT Aderivatives expressed in a baculovirus system. BoNT A td: toxicderivative of BoNT A. BoNT A ad: atoxic derivative of BoNT A. BoNT Agfpd: green fluorescent protein (GFP) derivative of BoNT A. Furthermodifications introduced into the td sequence depicted in FIG. 3 includethe introduction of a signal sequence and a hexahistidine tag in frontof the first native methionine for affinity purification. The differencebetween td and ad is a single amino acid substitution, E224>A, in theactive center of toxin's catalytic domain. To create BoNT A gfpd, aminoacids Tyr₁₀-Leu₄₁₆ of the native toxin's minimal catalytic domain weresubstituted with GFP. White and black arrows represent secretase andenterokinase cleavage sites, respectively.

FIG. 6 shows expression of BoNT A derivatives in a baculovirus system byWestern blot, probed with polyclonal antibodies raised againstfull-length BoNT A toxoid. Samples were treated with β-mercaptoethanolbefore separation. Protein molecular weight standards are shown on theleft. Lane 1, 2, 3, and 4: conditioned media from Sf9 cells infectedwith empty bacmid (Lane 1), or recombinant bacmids derived frompFBSBoNTA (Lane 2), pFBSBoNTAME224A (Lane 3) or pFBSGFPBoNTAHC (Lane 4).Lane 5 is native BoNT A as a positive control. Lanes 6, 7, 8, and 9:eluate after Ni-NTA affinity purification of conditioned media from Sf9cells transfected with empty bacmid (Lane 6), or recombinant bacmidsderived from pFBSBoNTA (Lane 7), pFBSBoNTAME224A (Lane 8), orpFBSGFPBONTAHC (Lane 9).

FIGS. 7A-B illustrate the concentration of recombinant enterokinase(rEK) required to effect complete cleavage of BoNT A toxic derivative(td) propeptide. FIG. 7A shows 8% PAGE stained with Coomassie G-250.FIG. 7B shows a Western blot of the gel in FIG. 7A, probed withpolyclonal antibodies raised against full-length BoNT A toxioid. Sampleswere treated with β-mercaptoethanol before the separation. Proteinmolecular weight standards are shown on the left. Different amounts ofrEK were added to 1 μg of BoNT A td in rEK cleavage buffer and incubatedat 20° C. for 8 hours. 10% of each reaction mixture was loaded per lane.The number of rEK units added per 1 μg of BoNT A td were: no rEK added(Lane 1); 0.05 U of rEK (Lane 2); 0.1 U of rEK (Lane 3); 0.25 U of rEK(Lane 4); 0.5 U of rEK (Lane 5). Lane 6 is the positive control, with0.1 μg of native BoNT A. The recombinant light chain is larger than thecontrol because of construct design.

FIGS. 8A-D show selected features of the recombinant BoNT A derivativesillustrating their native disulfide bonding (FIGS. 8A and 8B), and theuse of a signal sequence to increase secretion of the toxin derivativeinto the culture medium (FIGS. 8C and 8D). FIGS. 8A and 8B show PAGE ofthe indicated BoNT derivatives run on 10% PAGE gels, followed by Westernblotting using polyclonal antibodies raised against full-length BoNT Atoxioid. A protein molecular weight ladder is shown on the left. In FIG.8A, the PAGE was run under non-reducing conditions before transfer tothe nitrocellulose. In FIG. 8B, samples were treated withβ-mercaptoethanol and run under reducing conditions before transfer tothe nitrocellulose for Western blotting. Lane 1: Positive control,purified native BoNT A; Lane 2: BoNT A td cleaved with rEK; Lane 3: BoNTA ad cleaved with rEK; Lane 4: BoNT A gfpd cleaved with rEK. FIGS. 8Cand 8D are fluorescent images of the adherent layer of Sf9 cells(2·10⁵/cm²) in the SF 900 II medium at 12 hours post-infection (MOI˜0.1)with recombinant baculovirus expressing BoNT A gfpd containing thesignal peptide for secretion (FIG. 8C), or the control recombinantbaculovirus expressing GFP without added signal peptide (FIG. 8D).Emission wavelength 508 nm, magnification factor ×200, exposure time 0.1sec.

FIG. 9 is a BoNT A td purification table of 8% PAGE stained withCoomassie G-250. Samples were separated in the presence ofβ-mercaptoethanol. Lane 1: concentrated and dialyzed Sf9 medium, loadedon DEAE Sepharose; Lane 2: 100 mM NaCl eluate from DEAE Sepharose; Lane3: 200 mM NaCl eluate from MonoS column; Lane 4: 60 mM imidazole eluatefrom Ni-NTA agarose; Lane 5: material, eluted from the FPLCgel-filtration column; Lane 6: material, eluted from the FPLCgel-filtration column and digested with rEK; Lane 7: positive control,purified native BoNT A. Protein molecular weight ladder is shown on theright.

FIGS. 10A-B illustrate a transcytosis assay for polarized cells. Humangut epithelial cells (T-84) or canine kidney cells (MDCK) will be grownsubject to conditions that promote differentiation and polarization ofthe cell monolayer (FIG. 10A). An example of a polarized cellillustrating orientation of the apical membrane toward the top(accessible to medium in the insert) and the basal membrane orientedtoward the bottom (accessible to medium in the well) (FIG. 10B). Cellswill be grown on polycarbonate membranes coated with collagen inTranswell® porous bottom inserts. The inserts suspend the cell monolayerabove the bottom of the well, enabling cells to feed from the top andthe bottom, and to be exposed to toxin from the top and the bottom.Cultures grown in this manner differentiate into a polarized membranewith tight junctions.

FIGS. 11A-C illustrate the amino acid sequences of nine BoNT A chimericproteins containing SNARE motif peptides substituted for alpha-helixdomains in the light chain region aligned against the BoNT A ad protein(SEQ ID NO: 8). Chimera 1 (SEQ ID NO: 15) contains the full-lengthsequence of BoNT A ad with three SNARE motif peptides substituting lightchain alpha-helix 1. Chimera 2 (SEQ ID NO: 16) contains the full-lengthsequence of BoNT A ad with two SNARE motif peptides substituting lightchain alpha-helix 4. Chimera 3 (SEQ ID NO: 17) contains the full-lengthsequence of BoNT A ad with five SNARE motif peptides substituting lightchain alpha-helices 1 and 4. Chimera 4 (SEQ ID NO: 18) contains thefull-length sequence of BoNT A ad with three SNARE motif peptidessubstituting light chain alpha-helices 4 and 5. Chimera 5 (SEQ ID NO:19) contains the full length sequence of BoNT A ad with six SNARE motifpeptides substituting light chain alpha-helices 1, 4, and 5. Chimera 6(SEQ ID NO: 20) contains the full length sequence of BoNT A ad with fourSNARE motif peptides substituting light chain alpha-helices 4, 5, and 6.Chimera 7 (SEQ ID NO: 21) contains the full length sequence of BoNT A adwith five SNARE motif peptides substituting light chain alpha-helices 4,5, 6, and 7. Chimera 8 (SEQ ID NO: 22) contains the full length sequenceof BoNT A ad with seven SNARE motif peptides substituting light chainalpha-helices 1, 4, 5, and 6. Chimera 9 (SEQ ID NO: 23) contains thefull length sequence of BoNT A ad with eight SNARE motif peptidessubstituting light chain alpha-helices 1, 4, 5, 6, and 7.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to an isolated Clostridialneurotoxin propeptide. The propeptide has a light chain region, a heavychain region, where the light and heavy chain regions are linked by adisulfide bond, and an intermediate region connecting the light andheavy chain regions. The intermediate region has a highly specificprotease cleavage site which has three or more specific adjacent aminoacid residues that are recognized by the highly specific protease inorder to enable cleavage.

In a preferred embodiment, the isolated Clostridial neurotoxinpropeptide is from Clostridium botulinum. Clostridium botulinum hasmultiple serotypes (A-G). Although the Clostridial neurotoxinpropeptides of the present invention may be from any of the Clostridiumbotulinum serotypes, preferable serotypes are serotype A, serotype B,and serotype E.

Common structural features of the wild-type Clostridium botulinumneurotoxin propeptides are shown in FIG. 1. These structural featuresare illustrated using BoNT A propeptide as an example, and aregeneralized among all Clostridium botulinum serotypes. BoNT A propeptidehas two chains, a light chain (“LC”) of Mr ˜50,000 and a heavy chain(“HC”) of Mr ˜100,000, linked by a disulfide bond between Cys₄₂₉ andCys₄₅₃. As illustrated in FIG. 1, all seven BoNT serotype propeptideshave a light chain region and a heavy chain region linked by a disulfidebond. Two essential Cys residues, one adjacent to the C-terminus of thelight chain, and a second adjacent to the N-terminus of the heavy chainare present in all seven BoNT serotypes. These two Cys residues form thesingle disulfide bond holding the HC and LC polypeptides together in themature neurotoxin. This disulfide bond enables the mature neurotoxin toaccomplish its native physiological activities by permitting the HC andLC to carry out their respective biological roles in concert. Thedisulfide bond between HC and LC polypeptides in all seven serotypes isillustrated in FIG. 1 by the solid line joining the involved Cysresidues. The outlined box in FIG. 1 illustrates the intermediate regiondefined by amino acid residues Lys₄₃₈-Lys₄₄₈ of BoNT A. Thisintermediate region identifies the amino acids eliminated duringmaturation of wild-type BoNT A, and believed to be excised by a proteaseendogenous to the host microorganism. This cleavage event, describedinfra, generates the biologically active BoNT HC-LC dimer. The outlinedamino acid residues in FIG. 1, representing amino acid residues numberedapproximately in the 420 to 450 range for all seven BoNT serotypes, canbe considered as a region “non-essential” to the toxins' physiologicalactivity and, therefore, represents targets for directed mutagenesis inall seven BoNT serotypes.

All seven BoNT serotypes contain Lys or Arg residues in the intermediateregion defined by the box in FIG. 1 which make the propeptidessusceptible to activation by trypsin. Native BoNT A propeptide recoveredfrom young bacterial cultures can be activated by trypsinolysis, withproduction of intact, S—S bound light and heavy chain. Though multipleadditional trypsin-susceptible sites are present in the propeptides,they are resistant to proteolysis due to their spatial positions withinthe native toxin molecule (Dekleva et al., “Nicking of Single ChainClostridium botulinum Type A Neurotoxin by an Endogenous Protease,”Biochem. Biophys. Res. Commun. 162:767-772 (1989); Lacy et al., “CrystalStructure of Botulinum Neurotoxin Type A and Implications for Toxicity,”Nat. Struct. Biol. 5:898-902 (1998), which are hereby incorporated byreference in their entirety). A second site in the native propeptide ofseveral BoNT serotypes can be susceptible to trypsin cleavage whensubjected to higher enzyme concentrations or incubation times (Chaddocket al., “Expression and Purification of Catalytically Active, Non-ToxicEndopeptidase Derivatives of Clostridium botulinum Toxin Type A,”Protein Expr. Purif. 25:219-228 (2002), which is hereby incorporated byreference in its entirety). This trypsin-susceptible site is located inthe region adjacent to the toxin receptor binding domain. This region ofthe HC peptide is found to be exposed to solvent in BoNT serotypes forwhich information is available on their 3-D crystal structure (Lacy etal., “Crystal Structure of Botulinum Neurotoxin Type A and Implicationsfor Toxicity,” Nat. Struct. Biol. 5:898-902 (1998); Swaminathan et al.,“Structural Analysis of the Catalytic and Binding Sites of Clostridiumbotulinum Neurotoxin B,” Nat. Struct. Biol. 7:693-699 (2000), which arehereby incorporated by reference in their entirety).

In a preferred embodiment, the propeptide of the present invention hasan intermediate region connecting the light and heavy chain regionswhich has a highly specific protease cleavage site and nolow-specificity protease cleavage sites. For purposes of the presentinvention, a highly specific protease cleavage site has three or morespecific adjacent amino acid residues that are recognized by the highlyspecific protease in order to permit cleavage (e.g., an enterokinasecleavage site). In contrast, a low-specificity protease cleavage sitehas two or less adjacent amino acid residues that are recognized by aprotease in order to enable cleavage (e.g., a trypsin cleavage site).

In all seven BoNT serotypes, the amino acid preceding the N-terminus ofthe heavy chain is a Lys or Arg residue which is susceptible toproteolysis with trypsin. This trypsin-susceptible site can be replacedwith a five amino acid enterokinase cleavage site (i.e., DDDDK (SEQ IDNO: 24)) upstream of the heavy chain's N-terminus, as illustrated forthe seven serotypes in FIG. 2. This modification enables standardizationactivation with enterokinase. In serotypes A and C, additional Lysresidues within this region are mutated to either Gln or His, therebyeliminating additional trypsin-susceptible sites which might result inundesirable non-specific activation of the toxin. Trypsin-susceptiblerecognition sequences also occur upstream of the heavy chain'sreceptor-binding domain in serotypes A, E, and F. This region'ssusceptibility to proteolysis is consistent with its exposure to solventin the toxin's 3-D structure, as shown by X-ray crystallographyanalysis. Therefore, in serotypes A, E, and F, the susceptible residuesare modified to Asn (FIG. 2). Signal peptides and N-terminal affinitytags are also preferably introduced, as required, to enable secretionand recovery.

In a preferred embodiment, the isolated Clostridial neurotoxinpropeptide of the present invention has light and heavy chain regionswhich are not truncated.

As described in greater detail infra, the isolated Clostridialneurotoxin propeptide of the present invention may include a disablingmutation in an active metalloprotease site of the propeptide. The aminoacid residues constituting the minimal catalytic domain of the lightchain of the propeptide are illustrated in FIG. 1 and FIG. 2 byhatching. Specific amino acid residues constituting the active site ofthe catalytic domain of the metalloprotease are marked by stars in FIG.1 and FIG. 2.

The Clostridial neurotoxin propeptide of the present invention may alsopossess a non-native motif in the light chain region that is capable ofinactivating light chain metalloprotease activity in a toxic Clostridialneurotoxin. Suitable non-native motifs capable of inactivating lightchain metalloprotease activity of a toxic Clostridial neurotoxininclude, without limitation, SNARE motifs, metalloprotease inhibitormotifs, such as those present in the protein family known as TissueInhibitors of Metalloprotease (TIMP) (Mannello et al., “MatrixMetalloproteinase Inhibitors as Anticancer Therapeutics,” Curr. CancerDrug Targets 5:285-298 (2005); Emonard et al., “Regulation of MatrixMetalloproteinase (MMP) Activity by the Low-Density LipoproteinReceptor-Related Protein (LRP). A New Function for an ‘Old Friend,’”Biochimie 87:369-376 (2005); Maskos, “Crystal Structures of MMPs inComplex with Physiological and Pharmacological Inhibitors,” Biochimie87:249-263 (2005), which are hereby incorporated by reference in theirentirety), zinc chelating motifs based on suitably positioned sulfhydryl(preferably methionine) and acidic amino acids which become exposed uponbinding of the chimeric antagonist to the active LC metalloprotease, andpeptide motifs corresponding to the cleavage site on the substrate of LCmetalloproteases, including transition state analogs of said cleavagesite (Sukonpan et al., “Synthesis of Substrates and Inhibitors ofBotulinum Neurotoxin Type A Metalloprotease,” J. Peptide Res. 63:181-193(2004); Hayden et al., “Discovery and Design of Novel Inhibitors ofBotulinus Neurotoxin A: Targeted ‘Hinge’ Peptide Libraries,” Journal ofApplied Toxicology 23:1-7 (2003); Oost et al., “Design and Synthesis ofSubstrate-Based Inhibitors of Botulinum Neurotoxin Type BMetalloprotease,” Biopolymers (Peptide Science) 71:602-619 (2003), whichare hereby incorporated by reference in its entirety).

SNARE motif peptides have been shown to prevent cleavage of synapticcomplex components in Aplysia neurons (Rosetto et al., “SNARE Motif andNeurotoxins,” Nature 372:415-416 (1994), which is hereby incorporated byreference in its entirety). SNARE motif peptides are common to thesubstrate binding site of known BoNT serotypes, and have been shown toinhibit the toxic LC when injected into BoNT-affected neurons (Rosettoet al., “SNARE Motif and Neurotoxins,” Nature 372:415-416 (1994), whichis hereby incorporated by reference in its entirety).

In a preferred embodiment, the Clostridial neurotoxin propeptide lightchain region has one or more non-native motifs (e.g., SNARE motifpeptides), which replace surface alpha-helix domains of the nativepropeptide. Seven surface alpha-helix domains in the light chain regionof Clostridium botulinum serotypes are identified in FIG. 11.

A variety of Clostridial neurotoxin propeptides with light chain regionscontaining non-native motifs (e.g., SNARE motif peptides) in place ofsurface alpha-helix domains can be created. As described in greaterdetail below, these non-native motif bearing propeptides are generatedby altering the nucleotide sequences of nucleic acids encoding theClostridial neurotoxin propeptides.

Another aspect of the present invention relates to an isolated nucleicacid molecule encoding an isolated Clostridial neurotoxin propeptide ofthe present invention.

Nucleic acid molecules encoding full-length toxic Clostridialneurotoxins are well known in the art (See e.g., GenBank Accession Nos.M81186 (BONT B); D90210 (BoNT C); S49407 (BoNT D); D90210 (BoNT E);X81714 (BONT F); and X74162 (BONT G)).

Nucleic acid molecules of the present invention preferably encode theamino acid sequences of FIG. 2. In particular, the nucleic acidmolecules of the present invention are modified from the wild type BoNTserotype sequences to have one or more characteristics selected from thegroup consisting of a mutation which renders the encoded propeptideresistant to low-specificity proteolysis, one or more silent mutationsthat inactivate putative internal DNA regulatory elements, and one ormore unique restriction sites. In particular, and as illustrated foreach BoNT serotype in FIG. 2, mature neurotoxin stability and yield areoptimized by site-directed mutation of residues within the intermediateregion of the propeptide, thereby reducing the propeptides'susceptibility to non-specific proteolysis and poisoning of the hostorganism used for expression by the mature neurotoxin. Also, silentmutations are introduced into DNA regulatory elements that can affectRNA transcription or expression of the Clostridial neurotoxin propeptidein the system of choice. In addition, unique endonuclease restrictionsites are introduced to enable creation of chimeric proteins.

A nucleic acid molecule of the present invention may also have adisabling mutation in a region encoding an active metalloprotease siteof the propeptide, as described supra.

A nucleic acid molecule of the present invention may also have amutation in a region encoding the light chain region, such that thenucleic acid molecule encodes, in the light chain region, a non-nativemotif capable of inactivating light chain metalloprotease activity in atoxic clostridial neurotoxin. Suitable non-native motifs are describedsupra.

A further aspect of the present invention relates to an expressionsystem having a nucleic acid molecule encoding an isolated Clostridialneurotoxin propeptide of the present invention in a heterologous vector.

Yet another aspect of the present invention relates to a host cellhaving a heterologous nucleic acid molecule encoding an isolatedClostridial neurotoxin propeptide of the present invention.

Still another aspect of the present invention relates to a method ofexpressing a recombinant physiologically active Clostridial neurotoxinof the present invention. This method involves providing a nucleic acidconstruct having a nucleic acid molecule encoding an isolatedClostridial neurotoxin propeptide of the present invention. The nucleicacid construct has a heterologous promoter operably linked to thenucleic acid molecule and a 3′ regulatory region operably linked to thenucleic acid molecule. The nucleic acid construct is then introducedinto a host cell under conditions effective to express thephysiologically active Clostridial neurotoxin.

In a preferred embodiment, the expressed neurotoxin is contacted with ahighly specific protease under conditions effective to effect cleavageat the intermediate region. Preferably, the intermediate region of theClostridial neurotoxin propeptide is not cleaved by proteases endogenousto the expression system or the host cell.

Expression of a Clostridial neurotoxin of the present invention can becarried out by introducing a nucleic acid molecule encoding aClostridial neurotoxin propeptide into an expression system of choiceusing conventional recombinant technology. Generally, this involvesinserting the nucleic acid molecule into an expression system to whichthe molecule is heterologous (i.e., not normally present). Theintroduction of a particular foreign or native gene into a mammalianhost is facilitated by first introducing the gene sequence into asuitable nucleic acid vector. “Vector” is used herein to mean anygenetic element, such as a plasmid, phage, transposon, cosmid,chromosome, virus, virion, etc., which is capable of replication whenassociated with the proper control elements and which is capable oftransferring gene sequences between cells. Thus, the term includescloning and expression vectors, as well as viral vectors. Theheterologous nucleic acid molecule is inserted into the expressionsystem or vector in proper sense (5′→3′) orientation and correct readingframe. The vector contains the necessary elements for the transcriptionand translation of the inserted Clostridial neurotoxin propeptide-codingsequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference in its entirety, describes the production of expressionsystems in the form of recombinant plasmids using restriction enzymecleavage and ligation with DNA ligase. These recombinant plasmids arethen introduced by means of transformation and replicated in unicellularcultures including prokaryotic organisms and eukaryotic cells grown intissue culture.

Recombinant genes may also be introduced into viruses, includingvaccinia virus, adenovirus, and retroviruses, including lentivirus.Recombinant viruses can be generated by transfection of plasmids intocells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/− (see “Stratagene Cloning Systems” Catalog (1993) fromStratagene, La Jolla, Calif., which is hereby incorporated by referencein its entirety), pQE, pIH821, pGEX, pFastBac series (Invitrogen), pETseries (see F. W. Studier et. al., “Use of T7 RNA Polymerase to DirectExpression of Cloned Genes,” Gene Expression Technology Vol. 185 (1990),which is hereby incorporated by reference in its entirety), and anyderivatives thereof. Recombinant molecules can be introduced into cellsvia transformation, particularly transduction, conjugation,mobilization, or electroporation. The DNA sequences are cloned into thevector using standard cloning procedures in the art, as described bySambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsLaboratory, Cold Springs Harbor, N.Y. (1989), which is herebyincorporated by reference in its entirety.

A variety of host-vector systems may be utilized to express theClostridial neurotoxin propeptide-encoding sequence in a cell.Primarily, the vector system must be compatible with the host cell used.Host-vector systems include but are not limited to the following:bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA;microorganisms such as yeast containing yeast vectors; mammalian cellsystems infected with virus (e.g., vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g., baculovirus); and plantcells infected by bacteria. The expression elements of these vectorsvary in their strength and specificities. Depending upon the host-vectorsystem utilized, any one of a number of suitable transcription andtranslation elements can be used.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (“mRNA”)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eukaryotic promotersdiffer from those of prokaryotic promoters. Furthermore, eukaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a prokaryotic system, and, further, prokaryoticpromoters are not recognized and do not function in eukaryotic cells.

Similarly, translation of mRNA in prokaryotes depends upon the presenceof the proper prokaryotic signals which differ from those of eukaryotes.Efficient translation of mRNA in prokaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expressionsee Roberts and Lauer, Methods in Enzymology, 68:473 (1979), which ishereby incorporated by reference in its entirety.

Promoters vary in their “strength” (i.e., their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the PH promoter, T7 phage promoter, lacpromoter, trp promoter, recA promoter, ribosomal RNA promoter, the P_(R)and P_(L) promoters of coliphage lambda and others, including but notlimited, to lacUV5, ompF, bla, lpp, and the like, may be used to directhigh levels of transcription of adjacent DNA segments. Additionally, ahybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operons, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in prokaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ tothe initiation codon (ATG) to provide a ribosome binding site. Thus, anySD-ATG combination that can be utilized by host cell ribosomes may beemployed. Such combinations include but are not limited to the SD-ATGcombination from the cro gene or the N gene of coliphage lambda, or fromthe E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATGcombination produced by recombinant DNA or other techniques involvingincorporation of synthetic nucleotides may be used.

Depending on the vector system and host utilized, any number of suitabletranscription and/or translation elements, including constitutive,inducible, and repressible promoters, as well as minimal 5′ promoterelements may be used.

The Clostridial neurotoxin-encoding nucleic acid, a promoter molecule ofchoice, a suitable 3′ regulatory region, and if desired, a reportergene, are incorporated into a vector-expression system of choice toprepare a nucleic acid construct using standard cloning procedures knownin the art, such as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Third Edition, Cold Spring Harbor: Cold Spring HarborLaboratory Press, New York (2001), which is hereby incorporated byreference in its entirety.

The nucleic acid molecule encoding a Clostridial neurotoxin is insertedinto a vector in the sense (i.e., 5′→3′) direction, such that the openreading frame is properly oriented for the expression of the encodedClostridial neurotoxin propeptide under the control of a promoter ofchoice. Single or multiple nucleic acids may be ligated into anappropriate vector in this way, under the control of a suitablepromoter, to prepare a nucleic acid construct.

Once the isolated nucleic acid molecule encoding the Clostridialneurotoxin propeptide has been inserted into an expression vector, it isready to be incorporated into a host cell. Recombinant molecules can beintroduced into cells via transformation, particularly transduction,conjugation, lipofection, protoplast fusion, mobilization, particlebombardment, or electroporation. The DNA sequences are incorporated intothe host cell using standard cloning procedures known in the art, asdescribed by Sambrook et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), which is hereby incorporated by reference in its entirety.Suitable hosts include, but are not limited to, bacteria, virus, yeast,fungi, mammalian cells, insect cells, plant cells, and the like.Preferable host cells of the present invention include, but are notlimited to, Escherichia coli, insect cells, and Pichia pastoris cells.

Typically, an antibiotic or other compound useful for selective growthof the transformed cells only is added as a supplement to the media. Thecompound to be used will be dictated by the selectable marker elementpresent in the plasmid with which the host cell was transformed.Suitable genes are those which confer resistance to gentamycin, G418,hygromycin, puromycin, streptomycin, spectinomycin, tetracycline,chloramphenicol, and the like. Similarly, “reporter genes” which encodeenzymes providing for production of an identifiable compound, or othermarkers which indicate relevant information regarding the outcome ofgene delivery, are suitable. For example, various luminescent orphosphorescent reporter genes are also appropriate, such that thepresence of the heterologous gene may be ascertained visually.

In a preferred embodiment of the present invention, the expressedneurotoxin propeptide is contacted with a highly specific protease(e.g., enterokinase) under conditions effective to enable cleavage atthe intermediate region of the propeptide of the present invention.Preferably, the expressed neurotoxin propeptide has one or moredisulfide bridges.

Another aspect of the present invention relates to an isolated,physiologically active Clostridial neurotoxin produced by cleaving anisolated Clostridial neurotoxin propeptide of the present invention. Thepropeptide is cleaved at the highly specific protease cleavage site. Thelight and heavy chain regions are linked by a disulfide bond.

As discussed supra, Clostridial neurotoxins are synthesized as singlechain propeptides which are later activated by a specific proteolysiscleavage event, generating a dimer joined by a disulfide bond. Thesestructural features can be illustrated using BoNT A as an example, andare generally applicable to all Clostridium botulinum serotypes. Themature BoNT A is composed of three functional domains of Mr ˜50,000(FIG. 3A), where the catalytic function responsible for toxicity isconfined to the light chain (residues 1-437), the translocation activityis associated with the N-terminal half of the heavy chain (residues448-872), and cell binding is associated with its C-terminal half(residues 873-1,295) (Johnson, “Clostridial Toxins as TherapeuticAgents: Benefits of Nature's Most Toxic Proteins,” Annu. Rev. Microbiol.53:551-575 (1999); Montecucco et al., “Structure and Function of Tetanusand Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995), whichare hereby incorporated by reference in their entirety).

Optimized expression and recovery of recombinant neurotoxins for BoNTserotypes in a native and physiologically active state is achieved bythe introduction of one or more alterations to the nucleotide sequencesencoding the BoNT propeptides, as discussed supra. These mutations aredesigned to maximize yield of recombinant Clostridial neurotoxin, whileretaining the native toxins structure and biological activity.

Isolated, full-length Clostridial neurotoxins of the present inventionare physiologically active. This physiological activity includes, but isnot limited to, toxin immunogenicity, trans- and intra-cellulartrafficking, and cell recognition.

The mechanism of cellular binding and internalization of Clostridialtoxins is still poorly understood. No specific receptor has beenunambiguously identified, and the binding constants have not beencharacterized. The C-terminal portion of the heavy chain of allClostridial neurotoxins binds to gangliosides (sialic acid-containingglycolipids), with a preference for gangliosides of the G_(1b) series(Montecucco et al., “Structure and Function of Tetanus and BotulinumNeurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Montecucco, “How DoTetanus and Botulinum Toxins Bind to Neuronal Membranes?” TIBS11:314-317 (1986); and Van Heyningen et al., “The Fixation of TetanusToxin by Ganglioside,” J. Gen. Microbiol. 24:107-119 (1961), which arehereby incorporated by reference in their entirety). The sequenceresponsible for ganglioside binding has been identified for thestructurally similar TeNT molecule, and is located within the 34C-terminal amino acid residues of its heavy chain. BoNT A, B, C, E, andF share a high degree of homology with TeNT in this region (FIG. 1)(Shapiro et al., “Identification of a Ganglioside Recognition Domain ofTetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol.Chem. 272:30380-30386 (1997), which is hereby incorporated by referencein its entirety). Multiple types of evidence suggest the existence of atleast one additional component involved in the binding of Clostridialneurotoxins to neuronal membranes (Montecucco et al., “Structure andFunction of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys.28:423-472 (1995); Montecucco, “How Do Tetanus and Botulinum Toxins Bindto Neuronal Membranes?” TIBS 11:314-317 (1986), which are herebyincorporated by reference in their entirety). In two reports (Nishiki etal., “The High-Affinity Binding of Clostridium Botulinum Type BNeurotoxin to Synaptotagmin II Associated with GangliosidesG_(T1b)/G_(D1a) ,” FEBS Lett. 378:253-257 (1996); Dong et al.,“Synaptotagmins I and II Mediate Entry of Botulinum Neurotoxin B intoCells,” J. Cell Biol. 162:1293-1303 (2003), which are herebyincorporated by reference in their entirety), synaptotagmins wereidentified as possible candidates for the auxiliary BoNT B receptor, andsynaptotagmins I and II were implicated as neuronal receptors for BoNT G(Rummel et al., “Synaptotagmins I and II Act as Nerve Cell Receptors forBotulinum Neurotoxin G,” J. Biol. Chem. 279:30865-30870 (2004), which ishereby incorporated by reference in its entirety). However despite thestructural similarity in the putative receptor-binding domain ofClostridial neurotoxins, other toxin subtypes show no affinity forsynaptotagmins or synaptotagmin-related molecules. Lipid rafts (Herreroset al., “Lipid Rafts Act as Specialized Domains for Tetanus ToxinBinding and Internalization into Neurons,” Mol. Biol. Cell 12:2947-2960(2001), which is hereby incorporated by reference in its entirety) havebeen implicated as a specialized domain involved in TeNT binding andinternalization into neurons, but these domains are widely distributedon multiple cell types, and therefore cannot simply explain the highspecificity of the toxins for neurons.

Clostridial neurotoxins are internalized through the presynapticmembrane by an energy-dependent mechanism (Montecucco et al., “Structureand Function of Tetanus and Botulinum Neurotoxins,” Q. Rev. Biophys.28:423-472 (1995); Matteoli et al., “Synaptic Vesicle EndocytosisMediates the Entry of Tetanus Neurotoxin into Hippocampal Neurons,”Proc. Natl. Acad. Sci. USA 93:13310-13315 (1996); and Mukherjee et al.,“Endocytosis,” Physiol. Rev. 77:759-803 (1997), which are herebyincorporated by reference in their entirety), and rapidly appear invesicles where they are at least partially protected from degradation(Dolly et al., “Acceptors for Botulinum Neurotoxin Reside on Motor NerveTerminals and Mediate Its Internalization,” Nature 307:457-460 (1984);Critchley et al., “Fate of Tetanus Toxin Bound to the Surface of PrimaryNeurons in Culture: Evidence for Rapid Internalization,” J. Cell Biol.100:1499-1507 (1985), which are hereby incorporated by reference intheir entirety). The BoNT complex of light and heavy chains interactswith the endocytic vesicle membrane in a chaperone-like way, preventingaggregation and facilitating translocation of the light chain in afashion similar to the protein conducting/translocating channels ofsmooth ER, mitochondria, and chloroplasts (Koriazova et al.,“Translocation of Botulinum Neurotoxin Light Chain Protease through theHeavy Chain Channel,” Nat. Struct. Biol. 10:13-18 (2003), which ishereby incorporated by reference in its entirety). Acidification of theendosome is believed to induce pore formation, which allowstranslocation of the light chain to the cytosol upon reduction of theinterchain disulfide bond (Hoch et al., “Channels Formed by Botulinum,Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance toTranslocation of Proteins Across Membranes,” Proc. Natl. Acad. Sci. USA82:1692-1696 (1985), which is hereby incorporated by reference in itsentirety). Within the cytosol, the light chain displays azinc-endopeptidase activity specific for protein components of thesynaptic vesicle exocytosis apparatus. TeNT and BoNT B, D, F, and Grecognize VAMP/synaptobrevin. This integral protein of the synapticvesicle membrane is cleaved at a single peptide bond, which differs foreach neurotoxin. BoNT A, C, and E recognize and cleave SNAP-25, aprotein of the presynaptic membrane, at two different sites within thecarboxyl terminus. BoNT C also cleaves syntaxin, another protein of thenerve plasmalemma (Montecucco et al., “Structure and Function of Tetanusand Botulinum Neurotoxins,” Q. Rev. Biophys. 28:423-472 (1995); Suttonet al., “Crystal Structure of a SNARE Complex Involved in SynapticExocytosis at 2.4 Å Resolution,” Nature 395:347-353 (1998), which arehereby incorporated by reference in their entirety). The cleavage of anycomponent of the synaptic release machinery results in inhibition ofacetylcholine release, ultimately leading to neuromuscular paralysis.

In one embodiment of the present invention, the isolated Clostridialneurotoxin is toxic. The toxicity of Clostridial neurotoxins is a resultof a multi-step mechanism. From the circulation, BoNT targets thepre-synaptic membrane of neuromuscular junctions, where it isinternalized to directly exert its toxic effect on the peripheralnervous system (Dolly et al., “Acceptors for Botulinum Neurotoxin Resideon Motor Nerve Terminals and Mediate Its Internalization,” Nature307:457-460 (1984), which is hereby incorporated by reference in itsentirety). Toxicity at the neuromuscular junction involves neuronbinding; internalization into endocytic vesicles, similar to thoseinvolved in synaptic vesicle recycling; activation within an acidiccompartment to the proteolytically active toxin which then penetratesinto the neuronal cytoplasm; and target recognition and catalyticcleavage of substrates in the neuronal machinery for synaptic vesicleexocytosis.

In an alternative embodiment of the present invention, the isolatedClostridial neurotoxin is physiologically active and atoxic. Theendopeptidase activity responsible for Clostridial neurotoxin toxicityis believed to be associated with the presence of a HExxHxxH (SEQ ID NO:25) motif in the light chain, characteristic of metalloproteases (FIG.1). Mutagenesis of BoNT A light chain, followed by microinjection of thecorresponding mRNA into presynaptic cholinergic neurons of Aplysiacalifornica, allowed the minimal essential domain responsible fortoxicity to be identified (Kurazono et al., “Minimal Essential DomainsSpecifying Toxicity of the Light Chains of Tetanus Toxin and BotulinumNeurotoxin Type A,” J. Biol. Chem. 267:14721-14729 (1992), which ishereby incorporated by reference in its entirety). Site-directedmutagenesis of BoNT A light chain pinpointed the amino acid residuesinvolved in Zn²⁺ coordination, and formation of the activemetalloendoprotease core which cleaves SNAP-25 (Rigoni et al.,“Site-Directed Mutagenesis Identifies Active-Site Residues of the LightChain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun.288:1231-1237 (2001), which is hereby incorporated by reference in itsentirety). The three-dimensional structures of Clostridial neurotoxinsand their derivatives confirmed the mutagenesis results, and detailedthe spatial organization of the protein domains. For the BoNT Aholotoxin, crystal structure was obtained to a resolution of 3.3 Å (Lacyet al., “Crystal Structure of Botulinum Neurotoxin Type A andImplications for Toxicity,” Nat. Struct. Biol. 5:898-902 (1998), whichis hereby incorporated by reference in its entirety). The BoNT Bholotoxin crystal structure was determined at 1.8 and 2.6 Å resolution(Swaminathan et al., “Structural Analysis of the Catalytic and BindingSites of Clostridium Botulinum Neurotoxin B,” Nat. Struct. Biol.7:693-699 (2000), which is hereby incorporated by reference in itsentirety). Recently, a crystal structure for BoNT E catalytic domain wasdetermined to 2.1 Å resolution (Agarwal et al., “Structural Analysis ofBotulinum Neurotoxin Type E Catalytic Domain and Its Mutant Glu212>GlnReveals the Pivotal Role of the Glu212 Carboxylate in the CatalyticPathway,” Biochemistry 43:6637-6644 (2004), which is hereby incorporatedby reference in its entirety). The later study provided multipleinteresting structural details, and helps explain the complete loss ofmetalloendoproteolytic activity in the BoNT E LC E212>Q mutant. Theavailability of this detailed information on the relationship betweenthe amino acid sequence and biological activities of Clostridial toxinsenables the design of modified toxin genes with properties specificallyaltered for therapeutic goals.

Thus, in a preferred embodiment, the physiologically active and atoxicClostridial neurotoxin of the present invention has a disabling mutationin an active metalloprotease site.

The physiologically active and atoxic Clostridial neurotoxin of thepresent invention may also have a non-native motif (e.g., a SNARE motif)in the light chain region that is capable of inactivating light chainmetalloprotease activity in a toxic Clostridial neurotoxin. FIG. 11illustrates the sequences of nine chimeric proteins, which arephysiologically active and atoxic Clostridial neurotoxins containing atleast one non-native motif in the light chain region that is capable ofinactivating light chain metalloprotease activity in a toxic Clostridialneurotoxin. The non-native motifs are substituted for alpha-helixdomains. When present in the physiologically active and atoxicClostridial neurotoxin, the non-native protein motifs enable theneurotoxin to bind, inactivate, or otherwise mark the toxic light chainregion of a wild type Clostridial neurotoxin for elimination from thecytosol of neurotoxin-affected neurons. As such, a physiologicallyactive and atoxic Clostridial neurotoxin having a non-native motif inthe light chain region that is capable of inactiving light chainmetalloprotease activity in a toxic Clostridial neurotoxin is useful asan antidote to effectively target the cytoplasm of neurotoxin-affectedneurons. Administration of such antidotes is described in greater detailbelow.

Yet a further aspect of the present invention relates to a vaccine orantidote having an isolated, physiologically active, atoxic, Clostridialneurotoxin produced by cleaving an isolated Clostridial neurotoxinpropeptide of the present invention. The propeptide is cleaved at thehighly specific protease cleavage site. The light and heavy chainregions are linked by a disulfide bond.

Developing effective vaccines and antidotes against Clostridialneurotoxins requires the preservation of structural features importantto toxin trafficking and immunogenicity. From a practical perspective,this is most easily achieved by first producing recombinant moleculesthat retain the structural features and toxicity of native toxin,followed by selective modification to eliminate toxicity and introducetherapeutic utility. To achieve this goal, a versatile platform for thegenetic manipulation of Clostridial toxin genes and for their selectivemodification was developed (described infra). The genetic engineeringscheme can produce full-length toxic and atoxic derivatives of BoNT A,which retains important aspects of the wild toxin's native structure.This methodology can be generalized across the entire family ofClostridial neurotoxins because of their structural similarities (SeeFIGS. 1-2).

Thus, in a preferred embodiment, the vaccine or antidote of the presentinvention is a physiologically active and atoxic Clostridial neurotoxinfrom Clostridium botulinum, such as from Clostridium botulinum serotypesA-G. As described supra, the vaccine or antidote has the physiologicalactivity of a wild Clostridial neurotoxin, which activity includes, butis not limitated to, toxin immunogenicity, trans- and intra-cellulartrafficking, and cell recognition. The Clostridial neurotoxin of thevaccine or antidote is rendered atoxic by a mutation in its activemetalloprotease site, as described supra. Additional mutuations may beintroduced to ensure atoxicity and introduce new biological activities,while preserving systemic trafficking and cellular targeting of thevaccine or antidote. As has also been described, the vaccine or antidotemay possess non-native motifs in the light chain region that are capableof inactivating light chain metalloprotease activity in a toxicClostridial neurotoxin.

Atoxic Clostridial neurotoxins can be tested as candidate vaccines andantidotes to BoNT poisoning. Atoxic derivatives are created using theBoNT toxic derivative constructs developed under the methods describedinfra. Point mutations are introduced into the toxin's activemetalloprotease site to eliminate toxicity while maintaining nativetoxin structure, immunogenicity, trans- and intra-cellular trafficking,and cell recognition. Expression systems and purification schemes areoptimized as described infra. Derivatives found to completely lacktoxicity yet retain relevant biological activities of the native toxin,are evaluated for their potential as either vaccines or antidotes toBoNT poisoning. Parenteral routes of administration are tested first,followed by evaluation of oral and inhalational routes as applicable.Utility as a vaccine is determined by immunogenicity and challengestudies in mice. Utility as an antidote is first evaluated in vitro bytesting the ability of atoxic derivatives to prevent neuromuscularblockade in the mouse phrenic-nerve hemidiaphragm, and to inhibit nativetoxin trafficking in the transcytosis assay. Effective in vitroantagonists are tested as in vivo antidotes, and may be superior toantibody-based antidotes because they more effectively mimic nativetoxin absorption and trafficking pathways. Antidote effectiveness invivo is first evaluated using simultaneous dosing. Additional dosage andtiming parameters relevant to using antidotes under crisis situations isfurther evaluated for atoxic derivatives found to be effective whenadministered simultaneously with toxin. Using these procedures, a seriesof atoxic derivatives and fusion proteins are created and theirbiological activities systematically catalogued. The availability ofthese well characterized BoNT gene constructs and toxin derivativesenables the rational design of new anti-BoNT therapeutics. Dose-responseanalyses and challenge studies against active neurotoxin provide datathat allows the best candidate vaccines and antidotes to be selected forfurther development.

A further aspect of the present invention relates to method ofimmunizing a subject against toxic effects of a Clostridial neurotoxin.This method involves administering a vaccine of the present invention tothe subject under conditions effective to immunize the subject againsttoxic effects of Clostridial neurotoxin.

The subject administered the vaccine may further be administered abooster of the vaccine under conditions effective to enhanceimmunization of the subject.

Another aspect of the present invention relates to a method of treatinga subject for toxic effects of a Clostridial neurotoxin. This methodinvolves administering an antidote comprising an isolated,physiologically active, atoxic, Clostridial neurotoxin produced bycleaving the isolated Clostridial neurotoxin propeptide of the presentinvention to the subject under conditions effective to treat the subjectfor toxic effects of Clostridial neurotoxin.

A vaccine or antidote of the present invention can be administered to asubject orally, parenterally, for example, subcutaneously,intravenously, intramuscularly, intraperitoneally, by intranasalinstillation, or by application to mucous membranes, such as, that ofthe nose, throat, and bronchial tubes. The vaccine or antidote may beadministered alone or with suitable pharmaceutical carriers, and can bein solid or liquid form such as, tablets, capsules, powders, solutions,suspensions, or emulsions.

The vaccine or antidote of the present invention may be orallyadministered, for example, with an inert diluent, or with an assimilableedible carrier, or may be enclosed in hard or soft shell capsules, ormay be compressed into tablets, or may be incorporated directly with thefood of the diet. For oral therapeutic administration, the vaccine orantidote may be incorporated with excipients and used in the form oftablets, capsules, elixirs, suspensions, syrups, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compound in these compositions may, ofcourse, be varied and may conveniently be between about 2% to about 60%of the weight of the unit. The amount of active compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained. Preferred compositions according to the present inventionare prepared so that an oral dosage unit contains between about 1 and250 mg of active compound.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye, and flavoring such as cherry or orange flavor.

The vaccine or antidote may also be administered parenterally. Solutionsor suspensions can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solution,and glycols such as, propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The vaccine or antidote of the present invention may also beadministered directly to the airways in the form of an aerosol. For useas aerosols, the vaccine or antidote of the present invention insolution or suspension may be packaged in a pressurized aerosolcontainer together with suitable propellants, for example, hydrocarbonpropellants like propane, butane, or isobutane with conventionaladjuvants. The vaccine or antidote of the present invention also may beadministered in a non-pressurized form such as in a nebulizer oratomizer.

A further aspect of the present invention relates to a chimeric proteinhaving a first protein or protein fragment having a heavy chain regionof a Clostridial neurotoxin and a second protein or protein fragmentlinked to the first protein or protein fragment.

In a preferred embodiment, the second protein or protein fragment hastherapeutic functionality which can target specific steps in atrafficking pathway of the Clostridial neurotoxin.

BoNTs pass across epithelial surfaces without being destroyed or causinglocal toxicity. Passage across epithelia is believed to occur byspecific binding and transcytosis. The ability of intact BoNT A to passthough pulmonary epithelia and resist proteolytic inactivation wasdemonstrated in rat primary alveolar epithelial cells and inimmortalized human pulmonary adenocarcinoma (Calu-3) cells. The rate oftransport was greater in the apical-to-basolateral direction than in thebasolateral-to-apical direction, and it was blocked by serotype-specifictoxin antibodies (Park et al., “Inhalational Poisoning by BotulinumToxin and Inhalation Vaccination with Its Heavy-Chain Component,”Infect. Immun. 71:1147-1154 (2003), which is hereby incorporated byreference in its entirety).

The ability of Clostridial neurotoxins to pass undegraded throughepithelial barriers via transcytosis and to specifically target nervoustissue makes Clostridial neurotoxins useful in the development of oraland inhalational carriers for therapeutic agents that cannot normally bedelivered via these routes of administration, and as delivery vehicleswhich can specifically target the peripheral and central nervous system.

Still another aspect of the present invention relates to a treatmentmethod. This method involves contacting a patient with an isolated,physiologically active, toxic, Clostridial neurotoxin produced bycleaving an isolated Clostridial neurotoxin propeptide according to thepresent invention, under conditions effective to treat the patient.

By treatment, it is meant aesthetic treatment (See e.g., Carruthers etal., “Botulinum Toxin A in the Mid and Lower Face and Neck,” Dermatol.Clin. 22:151-158 (2004); Lang, “History and Uses of BOTOX (BotulinumToxin Type A),” Lippincotts Case Manag. 9:109-112 (2004); Naumann etal., “Safety of Botulinum Toxin Type A: A Systematic Review andMeta-Analysis,” Curr. Med. Res. Opin. 20:981-990 (2004); Vartanian etal., “Facial Rejuvenation Using Botulinum Toxin A: Review and Updates,”Facial Plast. Surg. 20:11-19 (2004), which are hereby incorporated byreference in their entirety) as well as therapeutic treatment (See e.g.,Bentsianov et al., “Noncosmetic Uses of Botulinum Toxin,” Clin.Dermatol. 22:82-88 (2004); Carruthers et al., “Botox: Beyond Wrinkles,”Clin. Dermatol. 22:89-93 (2004); Jankovic, “Botulinum Toxin In ClinicalPractice,” J. Neurol. Neurosurg. Psychiatry 75:951-957 (2004); Klein,“The Therapeutic Potential of Botulinum Toxin,” Dermatol. Surg.30:452-455 (2004); Schurch, “The Role of Botulinum Toxin in Neurology,”Drugs Today (Barc) 40:205-212 (2004), which are hereby incorporated byreference in their entirety).

Preferred treatment methods of the present invention include, but arenot limited to, dermatologic, gastroenterologic, genitourinaric, andneurologic treatment.

Dermatologic treatment includes, but is not limited to, treatment forRhtyiddess (wrinkles) (Sadick et al., “Comparison of Botulinum Toxins Aand B in the Treatment of Facial Rhytides,” Dermatol. Clin. 22:221-226(2004), which is hereby incorporated by reference in its entirety),including glabellar (Carruthers et al., “Botulinum Toxin type A for theTreatment of Glabellar Rhytides,” Dermatol. Clin. 22:137-144 (2004);Ozsoy et al., “Two-Plane Injection of Botulinum Exotoxin A in GlabellarFrown Lines,” Aesthetic Plast. Surg. 28:114-115 (2004); which are herebyincorporated by reference in their entirety), neck lines (Brandt et al.,“Botulinum Toxin for the Treatment of Neck Lines and Neck Bands,”Dermatol. Clin. 22:159-166 (2004), which is hereby incorporated byreference in its entirety), crows feet (Levy et al., “Botulinum Toxin A:A 9-Month Clinical and 3D In Vivo Profilometric Crow's Feet WrinkleFormation Study,” J. Cosmet. Laser Ther. 6:16-20 (2004), which is herebyincorporated by reference in its entirety), and brow contour (Chen etal., “Altering Brow Contour with Botulinum Toxin,” Facial Plast. Surg.Clin. North Am. 11:457-464 (2003), which is hereby incorporated byreference in its entirety). Other dermatologic treatment includestreatment for hypertrophic masateer muscles in Asians (Ahn et al.,“Botulinum Toxin for Masseter Reduction in Asian Patients,” Arch. FacialPlast. Surg. 6:188-191 (2004), which is hereby incorporated by referencein its entirety) and focal hyperhydrosis (Glogau, “Treatment ofHyperhidrosis with Botulinum Toxin,” Dermatol. Clin. 22:177-185, vii(2004), which is hereby incorporated by reference in its entirety),including axillary (“Botulinum Toxin (Botox) for AxillaryHyperhidrosis,” Med. Lett. Drugs Ther. 46:76 (2004), which is herebyincorporated by reference in its entirety) and genital (Lee et al., “ACase of Foul Genital Odor Treated with Botulinum Toxin A,” Dermatol.Surg. 30:1233-1235 (2004), which is hereby incorporated by reference inits entirety).

Gastroentologic treatment includes, but is not limited to, treatment foresophageal motility disorders (Achem, “Treatment of Spastic EsophagealMotility Disorders,” Gastroenterol. Clin. North Am. 33:107-124 (2004),which is hereby incorporated by reference in its entirety),pharyngeal-esophageal spasm (Bayles et al., “Operative Prevention andManagement of Voice-Limiting Pharyngoesophageal Spasm,” Otolaryngol.Clin. North Am. 37:547-558 (2004); Chao et al., “Management ofPharyngoesophageal Spasm with Botox,” Otolaryngol. Clin. North Am.37:559-566 (2004), which are hereby incorporated by reference in theirentirety), and anal fissure (Brisinda et al., “Botulinum Neurotoxin toTreat Chronic Anal Fissure: Results of a Randomized ‘Botox vs. Dysport’Controlled Trial,” Ailment Pharmacol. Ther. 19:695-701 (2004); Jost etal., “Botulinum Toxin A in Anal Fissure: Why Does it Work?” Dis. ColonRectum 47:257-258 (2004), which are hereby incorporated by reference intheir entirety).

Genitourinaric treatment includes, but is not limited to, treatment forneurogenic dysfunction of the urinary tract (“Botulinic Toxin inPatients with Neurogenic Dysfunction of the Lower Urinary Tracts,”Urologia July-August:44-48 (2004); Giannantoni et al., “IntravesicalResiniferatoxin Versus Botulinum-A Toxin Injections for NeurogenicDetrusor Overactivity: A Prospective Randomized Study,” J. Urol.172:240-243 (2004); Reitz et al., “Intravesical Therapy Options forNeurogenic Detrusor Overactivity,” Spinal Cord 42:267-272 (2004), whichare hereby incorporated by reference in their entirety), overactivebladder (Cruz, “Mechanisms Involved in New Therapies for OveractiveBladder,” Urology 63:65-73 (2004), which is hereby incorporated byreference in its entirety), and neuromodulation of urinary urgeincontinence (Abrams, “The Role of Neuromodulation in the Management ofUrinary Urge Incontinence,” BJU Int. 93:1116 (2004), which is herebyincorporated by reference in its entirety).

Neurologic treatment includes, but is not limited to, treatment fortourettes syndrome (Porta et al., “Treatment of Phonic Tics in Patientswith Tourette's Syndrome Using Botulinum Toxin Type A,” Neurol. Sci.24:420-423 (2004), which is hereby incorporated by reference in itsentirety) and focal muscle spasticity or dystonias (MacKinnon et al.,“Corticospinal Excitability Accompanying Ballistic Wrist Movements inPrimary Dystonia,” Mov. Disord. 19:273-284 (2004), which is herebyincorporated by reference in its entirety), including, but not limitedto, treatment for cervical dystonia (Haussermann et al., “Long-TermFollow-Up of Cervical Dystonia Patients Treated with Botulinum Toxin A,”Mov. Disord. 19:303-308 (2004), which is hereby incorporated byreference in its entirety), primary blepharospasm (Defazio et al.,“Primary Blepharospasm: Diagnosis and Management,” Drugs 64:237-244(2004), which is hereby incorporated by reference in its entirety),hemifacial spasm, post-stroke (Bakheit, “Optimising the Methods ofEvaluation of the Effectiveness of Botulinum Toxin Treatment ofPost-Stroke Muscle Spasticity,” J. Neurol. Neurosurg. Psychiatry75:665-666 (2004), which is hereby incorporated by reference in itsentirety), spasmodic dysphonia (Bender et al., “Speech Intelligibilityin Severe Adductor Spasmodic Dysphonia,” J. Speech Lang. Hear Res.47:21-32 (2004), which is hereby incorporated by reference in itsentirety), facial nerve disorders (Finn, “Botulinum Toxin Type A:Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol.3:133-137 (2004), which is hereby incorporated by reference in itsentirety), and Rasmussen syndrome (Lozsadi et al., “Botulinum Toxin AImproves Involuntary Limb Movements in Rasmussen Syndrome,” Neurology62:1233-1234 (2004), which is hereby incorporated by reference in itsentirety). Other neurologic treatments include treatment for amputationpain (Kern et al., “Effects of Botulinum Toxin Type B on Stump Pain andInvoluntary Movements of the Stump,” Am. J. Phys. Med. Rehabil.83:396-399 (2004), which is hereby incorporated by reference in itsentirety), voice tremor (Adler et al., “Botulinum Toxin Type A forTreating Voice Tremor,” Arch. Neurol. 61:1416-1420 (2004), which ishereby incorporated by reference in its entirety), crocodile tearsyndrome (Kyrmizakis et al., “The Use of Botulinum Toxin Type A in theTreatment of Frey and Crocodile Tears Syndrome,” J. Oral Maxillofac.Surg. 62:840-844 (2004), which is hereby incorporated by reference inits entirety), marginal mandibular nerve paralysis, and pain control(Cui et al., “Subcutaneous Administration of Botulinum Toxin A ReducesFormalin-Induced Pain,” Pain 107:125-133 (2004), which is herebyincorporated by reference in its entirety), including but not limited topain after mastectomy (Layeeque et al., “Botulinum Toxin Infiltrationfor Pain Control After Mastectomy and Expander Reconstruction,” Ann.Surg. 240:608-613 (2004), which is hereby incorporated by reference inits entirety) and chest pain of esophageal origin (Schumulson et al.,“Current and Future Treatment of Chest Pain of Presumed EsophagealOrigin,” Gastroenterol. Clin. North Am. 33:93-105 (2004), which ishereby incorporated by reference in its entirety). Another neurologictreatment amenable to the methods of the present invention is headache(Blumenfeld et al., “Botulinum Neurotoxin for the Treatment of Migraineand Other Primary Headache Disorders,” Dermatol. Clin. 22:167-175(2004), which is hereby incorporated by reference in its entirety).

The methods of the present invention are also suitable for treatment ofcerebral palsy (Balkrishnan et al., “Longitudinal Examination of HealthOutcomes Associated with Botulinum Toxin Use in Children with CerebralPalsy,” J. Surg. Orthop. Adv. 13:76-80 (2004); Berweck et al., “Use ofBotulinum Toxin in Pediatric Spasticity (Cerebral Palsy),” Mov. Disord.19:S162-S167 (2004); Pidcock, “The Emerging Role of TherapeuticBotulinum Toxin in the Treatment of Cerebral Palsy,” J. Pediatr.145:S33-S35 (2004), which are hereby incorporated by reference in theirentirety), hip adductor muscle dysfunction in multiple sclerosis (Wisselet al., “Botulinum Toxin Treatment of Hip Adductor Spasticity inMultiple Sclerosis,” Wien Klin Wochesnchr 4:20-24 (2001), which ishereby incorporated by reference in its entirety), neurogenic pain andinflammation, including arthritis, iatrogenic parotid sialocele(Capaccio et al., “Diagnosis and Therapeutic Management of latrogenicParotid Sialocele,” Ann. Otol. Rhinol. Laryngol. 113:562-564 (2004),which is hereby incorporated by reference in its entirety), and chronicTMJ displacement (Aquilina et al., “Reduction of a Chronic BilateralTemporomandibular Joint Dislocation with Intermaxillary Fixation andBotulinum Toxin A,” Br. J. Oral Maxillofac. Surg. 42:272-273 (2004),which is hereby incorporated by reference in its entirety). Otherconditions that can be treated by local controlled delivery ofpharmaceutically active toxin include intra-articular administration forthe treatment of arthritic conditions (Mahowald et al., “Long TermEffects of Intra-Articular BoNT A for Refractory Joint Pain,” AnnualMeeting of the American College of Rheumatology (2004), which is herebyincorporated by reference in its entirety), and local administration forthe treatment of joint contracture (Russman et al., “Cerebral Palsy: ARational Approach to a Treatment Protocol, and the Role of BotulinumToxin in Treatment,” Muscle Nerve Suppl. 6:S181-S193 (1997); Pucinelliet al., “Botulinic Toxin for the Rehabilitation of OsteoarthritisFixed-Flexion Knee Deformity,” Annual Meeting of the OsteoarthitisResearch Society International (2004), which are hereby incorporated byreference in their entirety). The methods of the present invention arealso suitable for the treatment of pain associated with variousconditions characterized by the sensitization of nociceptors and theirassociated clinical syndromes, as described in Bach-Rojecky et al.,“Antinociceptive Effect of Botulinum Toxin Type A In Rat Model ofCarrageenan and Capsaicin Induced Pain,” Croat. Med. J. 46:201-208(2005); Aoki, “Evidence for Antinociceptive Activity of Botulinum ToxinType A in Pain Management,” Headache 43 Suppl 1:S9-15 (2003); Kramer etal., “Botulinum Toxin A Reduces Neurogenic Flare But Has Almost NoEffect on Pain and Hyperalgesia in Human Skin,”J. Neurol. 250:188-193(2003); Blersch et al., “Botulinum Toxin A and the Cutaneous Nociceptionin Humans: A Prospective, Double-Blind, Placebo-Controlled, RandomizedStudy,” J. Neurol. Sci. 205:59-63 (2002), which are hereby incorporatedby reference in its entirety.

The methods and products of the present invention may be customized tooptimize therapeutic properties (See e.g., Chaddock et al., “RetargetedClostridial Endopeptidases: Inhibition of Nociceptive NeurotransmitterRelease In Vitro, and Antinociceptive Activity in In Vivo Models ofPain,” Mov. Disord. 8:S42-S47 (2004); Finn, “Botulinum Toxin Type A:Fine-Tuning Treatment of Facial Nerve Injury,” J. Drugs Dermatol.3:133-137 (2004); Eleopra et al., “Different Types of Botulinum Toxin inHumans,” Mov. Disord. 8:S53-S59 (2004); Flynn, “Myobloc,” Dermatol.Clin. 22:207-211 (2004); and Sampaio et al., “Clinical Comparability ofMarketed Formulations of Botulinum Toxin,” Mov. Disord. 8:S129-S136(2004), which are hereby incorporated by reference in their entirety).

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 SDS PAGE

Samples from all intermediate purification steps, as well as purerecombinant protein, were routinely separated and visualized on 8%separating polyacrylamide gels, according to Laemmli procedure (Laemmli,“Cleavage of Structural Proteins During the Assembly of the Head ofBacteriophage T4,” Nature 227:680-685 (1970), which is herebyincorporated by reference in its entirety). Protein bands werevisualized by Bio-Safe Coomassie G-250 Stain (Bio-Rad, Cat. #161-0786).

Example 2 Western Blotting

Samples for Western blot analysis were separated on 8%SDS-polyacrylamide gels. Followed by separation, proteins weretransferred to the Hybond-C nitrocellulose membrane (AmershamBiosciences, Cat.#RPN₃O₃C) in 1× Tris/Glycine buffer (Bio-Rad,Cat.#161-0734) supplemented with 20% methanol at 100 volts for 2 hours,4° C. After the transfer, membrane was rinsed in distilled water andprotein bands were visualized by staining with 0.2% Ponceau S in 1%acetic acid for 1 minute. Dye from the membrane was washed away in theTris-buffered saline/0.1% Tween-20 buffer, pH 7.5, followed byincubation of the membrane in the blocking reagent (5% non-fat powderedmilk in Tris-buffered saline/0.1% Tween-20 buffer, pH 7.5) for 16 hoursat 4° C. For immunodetection, membrane was incubated with primaryantibodies/immune serum at 1:7,000 dilution, in 0.5% non-fat milk inTris-buffered saline/0.1% Tween-20 buffer, pH 7.5 at room temperaturefor 2 hours. Membrane was washed (6×5 min) and incubated with secondaryantibody at 1:10,000 dilution at room temperature for 25 minutes. Afterthe series of additional washing (6×5 min), immunoreactive bands werevisualized using ECL (enhanced chemiluminescence) Plus Western BlottingReagent (Amersham Biosciences, Cat.#RPN2124) according to manufacturerinstructions. Hyperfilm ECL (Amersham Biosciences, Cat.#RPN1674K) wasused for autoradiography with the exposure time adequate to visualizechemiluminescent bands. The proteins were identified by comparison withthe positive controls and molecular weight protein standards.

Example 3 Evaluation of Recombinant Toxin Yield

The protein concentration of the purified recombinant protein fractionswere determined using the BCA Protein assay reagent (Pierce, Cat.#23225)with bovine serum albumin used as standard.

Example 4 In vitro Toxicity Assay on the Mouse PhrenicNerve-Hemidiaphragm Preparation

The toxicity of the various recombinant proteins is bioassayed on themouse phrenic nerve-hemidiaphragm preparation (Simpson et al.,“Isolation and Characterization of a Novel Human Monoclonal Antibodythat Neutralizes Tetanus Toxin,” J. Pharmacol. Exp. Ther. 254:98-103(1990), which is hereby incorporated by reference in its entirety).Tissues are excised and suspended in physiological buffer, aerated with95% O₂, 5% CO₂, and maintained at 35° C. The physiological solution hasthe following composition: 137 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1 mMMgSO₄, 24 mM NaHCO₃, 1 mM NaH₂PO₄, 11 mM D-glucose, and 0.01% gelatin.Phrenic nerves are stimulated continuously (1.0 Hz; 0.1-0.3 msecduration), and muscle twitch is recorded. Toxin-induced paralysis ismeasured as a 50% reduction in muscle twitch response to neurogenicstimulation.

Example 5 In Vitro Transcytosis Assay

Cells are grown on polycarbonate membranes with a 0.4 μm pore size inTranswell® porous bottom inserts (Corning-Costar) (FIG. 10) (Zweibaum etal., “Use of Cultured Cell Lines in Studies of Intestinal CellDifferentiation and Function,” In: Handbook of Physiology, Section 6:“The Gastrointestinal System,” Edited by Schulz et al., AmericanPhysiological Society, Bethesda, Vol. IV, 223-255; Dharmsathaphorn etal., “A Human Colonic Tumor Cell Line that Maintains VectorialElectrolyte Transport,” Am. J. Physiol. 246:G204-G208 (1984); andDharmsathaphorn et al., “Established Intestinal Cell Lines as ModelSystems for Electrolyte Transport Studies,” Methods Enzymol. 192:354-389(1990), which are hereby incorporated by reference in their entirety).The cell growth area within each insert is equivalent to 1 cm². Prior toseeding the cells, insert membranes are coated with 10 μg/cm² rat tailcollagen type I. Collagen stock solution (6.7 mg/ml) are prepared insterile 1% acetic acid and stored at 4° C. The collagen stock solutionis diluted as needed in ice cold 60% ethanol, and 150 μl of theresulting solution containing 10 μg of diluted collagen is added to eachwell (cm²).

The collagen solution is allowed to dry at room temperature overnight(ca. 18 hours). After drying, the wells are sterilized under UV lightfor one hour, followed by a preincubation with cell culture medium (30minutes). The preincubation medium is removed immediately prior toaddition of cells and fresh medium. Cells are plated in the Transwells®at confluent density. The volumes of medium added will be 0.5 ml to theupper chamber and 1.0 ml to the bottom chamber. Culture medium ischanged every two days. The cultures maintained in 12 well plates areallowed to differentiate a minimum of 10 days before use. The integrityof cell monolayers and formation of tight junctions is visualized bymonitoring the maintenance of a slightly higher medium meniscus in theinserts as compared to the bottom wells.

Formation of tight junctions is confirmed experimentally by assay of therate of [³H]-inulin diffusion from the top well into the bottom chamberor by measurement of transepithelial resistance across the monolayer.Transcytosis is assayed by replacement of medium, usually in the topwell, with an appropriate volume of medium containing variousconcentrations of [¹²⁵I]-labeled protein of interest. Iodination isperformed according to Park et al., “Inhalational Poisoning by BotulinumToxin and Inhalation Vaccination with Its Heavy-Chain Component,”Infect. Immun. 71:1147-1154 (2003), which is hereby incorporated byreference in its entirety. Transport of radiolabeled protein ismonitored by sampling the entire contents of opposite wells, which isusually the bottom wells. Aliquots (0.5 ml) of the sampled medium arefiltered through a Sephadex G-25 column, and 0.5 ml fractions arecollected. The amount of radioactivity in the fractions is determined ina γ-counter. The amount of transcytosed protein is normalized andexpressed as fmole/hr/cm². A minimum of two replicates per condition isincluded in each experiment, and experiments typically are reproduced atleast three times.

Example 6 In Vivo Toxicity Assay in Mice

The toxicity of proteins of interest are bioassayed in mice. Proteinsare diluted in phosphate buffered saline, including 1 mg/ml bovine serumalbumin, and injected intraperitoneally (i.p.) into animals. Theproteins are administered in a 100 μl aliquot of solution atconcentrations of 1-100 ng per animal (average weight 25 g). Any animalsthat survive exposure to the toxic derivatives are monitored for a totalof 2 weeks to detect any non-specific toxicity.

Example 7 The BoNT Substrate-Cleavage Assay

Engineered proteins are assayed for endoprotease activity using eithermouse brain synaptosomes and recombinant SNAP-25 for BoNT A and BoNT Eas the source of the substrate. Native or reduced proteins are incubatedwith 10 to 50 μg of synaptosomal membranes in reaction buffer containing50 mM HEPES, pH 7.1, 20 μM ZnCl₂, and 1% N-octyl-β-D-glucopyranoside.Reduced protein are prepared by incubation with DTT (20 mM; 1 hr; roomtemperature) in phosphate buffered saline. The cleavage reaction isinitiated by addition of engineered protein (200 nM final concentration)to substrate, and the reaction is allowed to proceed for 3 hours at 37°C. Endoprotease activity is assayed using Western blot analysis andanti-C-terminal SNAP-25 antibodies (StressGen) for immunodetection ofsubstrate. For visualization of SNAP-25, samples are separated on 16.5%Tris-tricine gels. After separation, proteins are transferred tonitrocellulose membranes (Micron Separations) in Tris-glycine transferbuffer at 50 volts for 1 hr. Blotted membranes are rinsed in distilledwater and stained for 1 min with 0.2% Ponceau S in 1% acetic acid.Following a brief rinse with distilled water, molecular weight markersand transferred proteins are visualized. Membranes are destained inphosphate buffered saline-Tween (pH 7.5; 0.1% Tween 20), then blockedwith 5% non-fat powdered milk in phosphate buffered saline-Tween for 1hr at room temperature. Subsequently, membranes are incubated in 0.5%milk with a 1:5,000 dilution of anti-SNAP-25 polyclonal antibody.Secondary antibody is used at 1:20,000 dilution. Membranes are washedagain (5×) and visualized using enhanced chemiluminescence(SuperSignal®West Pico, Pierce) according to manufacturer'sinstructions. Membranes are exposed to film (Hyperfilm ECL, AmershamBiosciences) for times adequate to visualize chemiluminescence bands.Peptides are identified by comparison with known standards. The BoNT Bsubstrate-cleavage assay is performed according to the publishedprotocol (Caccin et al., “VAMP/Synaptobrevin Cleavage by Tetanus andBotulinum Neurotoxins is Strongly Enhanced by Acidic Liposomes,” FEBSLett. 542:132-136 (2003), which is hereby incorporated by reference inits entirety).

Example 8 Cloning Procedures: Preparation of the DNA Template for PCR

Outlined in detail infra are the procedures used to engineer BoNT Aderivatives. A similar strategy for engineering all BoNT derivatives canbe carried out.

25 μg of the pure Clostridium botulinum type A (Hall strain) genomic DNAwas isolated from bacterial pellet separated from the 100 ml of theculture according to Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Plainview, N.Y.: Cold Spring Harbor LaboratoryPress (1989), which is hereby incorporated by reference in its entirety.DNA was precipitated and dissolved in 1×TE, pH 8.0, at concentration˜0.8 mg/ml.

Genomic DNA, isolated from the mixture of the anaerobic bacteria fromthe soil, was prepared according to the following protocol: 1000 g ofthe soil taken from Central Park, N.Y., were triturated in 2 liters ofDulbecco's phosphate-buffered saline (DPBS) (Invitrogen,Cat.#14190-144). Crude extract was filtered through Kimwipes EX-L wipes(Kimberly-Clark, Neenah, Wis.) and concentrated on a stirredultrafiltration cell (Millipore (Billerica, Mass.), Cat.#5123) withUltracel 100-KDa cutoff membrane (Millipore, Cat.#14432) to a finalvolume of 5 ml. Four liters of cooked meat medium (Difco (FranklinLakes, N.J.), Cat.#226720), prepared according to manufacturer'sprotocol were inoculated with 5 ml of concentrated soil extract. After168-hour incubation at 37° C. without agitation or aeration, a mixtureof anaerobic bacteria was separated from the supernatant bycentrifugation on Sorwall GS3 rotor (7000 rpm, 25 min., 4° C.) andprocessed for the isolation of the total genomic DNA on Qiagen(Valencia, Calif.) Genomic tips (Cat.#10262), with additional componentsalso purchased from Qiagen (Cat.#19060, Cat.#19133, Cat.#19101),according to manufacturer's protocol (Qiagen Genomic DNA Handbook). Fromthe cells recovered from 4 liters of the media on ten Qiagen Genomictips, 6 mg of the genomic DNA were isolated. DNA was precipitated anddissolved in 1×TE, pH 8.0 at concentration ˜1 mg/ml.

Example 9 PCR Amplification of BoNT DNA

25 μg of the mixed genomic DNA or 5 μg of the pure Clostridium botulinumtype A genomic DNA were used per one 100-μl PCR reaction setting.Reaction conditions were designed according to manufacturer's protocolssupplied with Platinum®Pfx polymerase (Invitrogen, Cat.#11708-021). Alloligonucleotides and linkers were designed according to the sequence ofbotulinum Neurotoxin type A cDNA obtained from Genebank (Accession #:M30196). Annealing temperatures were deduced from the structure of eachset of the oligonucleotides used for the PCR.

Example 10 Engineering of Non-Expression Vector pLitBoNTA, CarryingCoding Part of BoNT A td

Plasmid encoding botulinum Neurotoxin A light chain (pLitBoNTALC) wasobtained by the following protocol: The annealed phosphorylated linkers

CBA01: (SEQ ID NO: 26) 5′-pCTAGCATGCCATTTGTTAATAAACAATTTAATTATAAG andCBA02: (SEQ ID NO: 27) 5′-pGATCCTTATAATTAAATTGTTTATTAACAAATGGCATGwere subcloned into vector pcDNA3.1/Zeo(+) (Invitrogen, Cat.#V86020),pre-digested with the restriction endonucleases NheI and BamHI anddephosphorylated, resulting in plasmid pcDBoNTALC1. The 620 b.p. PCRproduct, obtained on genomic DNA as a template with the oligonucleotides

CBA03: (SEQ ID NO: 28)5′-TATCTGCAGGGATCCTGTAAATGGTGTTGATATTGCTTATATAAAAA TTCC and CBA04: (SEQID NO: 29) 5′-TATGAATTCACCGGTCCGCGGGATCTGTAGCAAATTTGCCTGCACCwas digested with the restriction endonucleases BamHI and EcoRI andsubcloned into pre-digested plasmid pcDBoNTALC1, resulting in plasmidpcDBoNTALC2. The 630 b.p. PCR product, obtained on genomic DNA as atemplate with the oligonucleotides

CBA05: (SEQ ID NO: 30)5′-TATACCGCGGTAACATTAGCACATGAACTTATACATGCTGGACATAG ATTATATG and CBA06:(SEQ ID NO: 31) 5′-CATAGAATTCAAACAATCCAGTAAAATTTTTTAGTTTAGTAAAATTCATATTATTAATTTCTGTATTTTGACC,was digested with the restriction endonucleases SacII and EcoRI andsubcloned into pre-digested plasmid pcDBoNTLC2, resulting in plasmidpcDBoNTLC3. The annealed phosphorylated linkers

CBA08: (SEQ ID NO: 32) 5′-pAATTCTATAAGTTGCTATGTGTAAGAGGGATAATACTAGTCACAC TCAATCT and CBA09: (SEQ IDNO: 33) 5′-pCTAGAGATTGAGTGTGACTAGTTATTATCCCTCTTACACATAGCAA CTTATAGwere subcloned into vector pcDBoNTLC3, pre-digested with the restrictionendonucleases EcoR and XbaI and dephosphorylated, resulting in plasmidpcDBoNTALC. The annealed phosphorylated linkers

CBA10: (SEQ ID NO: 34) 5′-pCGCGTTAGCCATAAATCTGGTTATAAGCGCGCGAGGTGTTAAGTGand CBA11: (SEQ ID NO: 35)5′-pCTAGCACTTAACACCTCGCGCGCTTATAACCAGATTTATGGCTAAwere subcloned into vector pLitmus38i (New England Biolabs,Cat.#N3538S), pre-digested with the restriction endonucleases MluI andNheI and dephosphorylated, resulting in plasmid pLit38iMod. The 1230b.p. DNA fragment, isolated from the plasmid pcDBoNALC after its digestwith restriction endonucleases NheI and ApaI was subcloned intopre-digested and dephosphorylated vector pLit38iMod, resulting inplasmid pLitBoNTALC.

Plasmid encoding botulinum Neurotoxin A heavy chain (pLitBoNTAHC) wasobtained by the following protocol: The 1450 b.p. PCR product obtainedon the genomic DNA as a template with the oligonucleotides

CBA12: (SEQ ID NO: 36)5′-AATCTGCAGCCACAGCTGTGGGGTACCTTAATTGGTCAAGTAGATAGATTAAAAGATAAAGTTAATAATACACTTAGTACAGATATACC and CBA13: (SEQ ID NO: 37)5′-ATTAGGGCCCTTAATTAAGCGGCCGCCTCGAGCTATTACAGTGGCCTTTCTCCCCATCCATCATCTACAGGAATAAATTCwas digested with restriction endonucleases ApaI and PstI and subclonedinto pre-digested and dephosphorylated vector pLitmus38i, resulting inplasmid pLitBoNTAHC1. Two PCR products, 490 b.p., obtained on thegenomic DNA as a template with the oligonucleotides

CBA14: (SEQ ID NO: 38) 5′-ATACTGCAGTCTAGACCAAGGATACAATGACGATGATGATAAGGCATTAAATGATTTATGTATCAAAGTTAATAATTGGG and CBA15: (SEQ ID NO: 39)5′-GCCTAAAAACATAGCCGCTTCGGTCGCTTTATTAACTTTCTTTACAT AGTCTGAAGand 720 b.p., obtained on genomic DNA as a template with theoligonucleotides

CBA16: (SEQ ID NO: 40)5′-TAATAAAGCGACCGAAGCGGCTATGTTTTTAGGCTGGGTAGAACAAT TAG and CBA17: (SEQID NO: 41) 5′-TATAGGGCCCCCTAGGGGTACCTCTATTATCATATATATACTTTAATAATGCATCTTTAAGACwere mixed with the molar ratio 1:1 and re-PCRed with oligonucleotidesCBA14 and CBA17, resulting in 1170 b.p. PCR product, which was digestedwith restriction endonucleases PstI and KpnI and subcloned intopre-digested and dephosphorylated vector pLitBoNTAHC1, leading toplasmid pLitBoNTAHC.

Plasmid pLitBoNTA, encoding the entire sequence of BoNT A was obtainedby ligating a 2615 b.p. DNA fragment from the vector pLitBoNTAHC,digested with restriction endonucleases XbaI and ApaI into pre-digestedand dephosphorylated vector pLitBoNTALC. The size of pLitBoNTA is 6712b.p. with 3900 b.p. of BoNT A coding sequence.

Example 11 Engineering Plasmid pETcBoNTA for the BoNT A td Expression inE. coli

pETCBoNTA was obtained by subcloning DNA fragment obtained after thedigest of pLitBoNTA vector with NheI and NotI into pre-digested anddephosphorylated expression vector pETcoco2 (Novagen (San Diego,Calif.), Cat.#71148-3) and resulted in 16,194 b.p. BoNT A td expressionvector pETCBoNTA.

Example 12 Engineering Donor Plasmid pFBSecBoNTA for the Expression ofBoNT A td in Insect Cells

pFBSecBoNTA was obtained by the following protocol: 112 b.p. PCRproduct, synthesized on plasmid pBac-3 (Novagen, Cat.#70088-3) witholigonucleotides

CBA 22: (SEQ ID NO: 42) 5′-TAAGCGCGCAGAATTCTCTAGAAT GCCCATGTTAAGCGCTATTGand CBA23: (SEQ ID NO: 43) 5′-TAAGCTAGCGTGATGGTGGTGATGATGGACCATGGCCand digested with restriction endonucleases BssHII and NheI wassubcloned into pre-digested and dephosphorylated vector pLitBoNTA,resulting in plasmid pLitSecBoNTA. DNA fragment, isolated frompLitSecBoNTA digested with BssHII and NotI was subcloned intopre-digested and dephosphorylated vector pFastBac™1 (Invitrogen,Cat.#10360-014), resulting in 8764 b.p. plasmid pFBSecBoNTA.

Example 13 Engineering the BoNT A Coding Sequence to Enable Expressionof Toxin Derivatives

The DNA template was obtained as either pure genomic DNA isolated fromClostridium botulinum type A cultures, or as mixed genomic DNA isolatedfrom anaerobic bacteria of soil. BoNT A DNA was amplified by PCR usingthe high fidelity Platinum Pfx polymerase (Invitrogen, Carlsbad,Calif.). The full-length coding sequence of BoNT A toxic derivative (td)was obtained after consecutive subcloning of five PCR fragments and twophosphorylated linkers into the modified vector pLitmus38i (New EnglandBiolabs, Beverly, Mass.), resulting in plasmid pLitBoNTA. This strategywas used to minimize infidelity during the PCR reaction and to enablethe introduction of targeted mutations and endonuclease restrictionsites for subsequent engineering of expressed toxin products.

The details of the final construct (td) in comparison with native BoNT A(wt) are shown in FIG. 3, illustrating new restriction sites, eliminatedalternative translation sites, and amino acids inserted or substituted.The construct encoding full-length BoNT A td was obtained by ligation ofthe DNA inserts from the plasmid encoding the toxin heavy chain (“HC”)into the plasmid encoding the toxin light chain (“LC”). Plasmid encodingthe LC of BoNT A td was generated by consecutive ligation of two PCRproducts and two phosphorylated linkers into vector pLitmus38i. Itcontains multiple unique restriction sites upstream from the 5′-end ofthe LC sequence, the unique endonuclease restriction site NheI upstreamfrom the first methionine codon, and endonuclease restriction sites forBamHI and EcoRI introduced by silent mutations flanking the minimalcatalytic domain (Kadkhodayan et al., “Cloning, Expression, and One-StepPurification of the Minimal Essential Domain of the Light Chain ofBotulinum Neurotoxin Type A,” Protein Expr. Purif 19:125-130 (2000),which is hereby incorporated by reference in its entirety) of theprotein at the codons for Lys₁₁ and Phe₄₂₅. Two additional mutationsencoding substitutions Lys₄₃₈>His and Lys₄₄₀>Gln were introduced tominimize non-specific proteolysis of the BoNT A td propeptide duringexpression. A unique restriction site for XbaI was introduced by silentmutation at the codon. Plasmid, encoding the HC of BoNT A td, wasgenerated by consecutive ligation of two PCR products into thepLitmus38i vector. First, the PCR product encoding the receptor-bindingdomain of BoNT A was subcloned into the vector pLitmus38i. Second, thePCR product encoding the toxin's translocation domain, obtained byre-PCR of two smaller PCR products was subcloned into plasmid encodingthe toxin's receptor binding domain. The final plasmid contains a uniqueXbaI site at the 5′-end of the coding sequence introduced by silentmutation of the codon Asp₄₄₃, mutation of Lys₄₄₄>Gln to minimizenon-specific proteolysis of the BoNT A td propeptide, insertion ofcodons for four aspartic acid residues between Asn₄₄₇ and Lys₄₄₈ tocreate an enterokinase cleavage site, four silent mutations at Ala₅₉₇,Thr₅₉₈, Glu₅₉₉, and Ala₆₀₀ to inactivate the putative internal DNAregulatory element, a unique KpnI site introduced at the codon forGly₈₂₉ by silent mutagenesis, and multiple unique restriction sites atthe 3′-end of the construct after the stop codon. DNA encoding theAla₅₉₇-Ala₆₀₀ sequence was mutated, because it contains an internalShine-Dalgarno sequence upstream from internal methionine codon whichcan result in co-translational contamination of recombinant proteinexpressed in E. coli (Lacy et al., “Recombinant Expression andPurification of the Botulinum Neurotoxin Type A Translocation Domain,”Protein Expr. Purif. 111: 195-200 (1997), which is hereby incorporatedby reference in its entirety), the initial choice for an expressionsystem to test.

A second full-length BoNT A gene derivative was designed to render theBoNT A atoxic (ad, atoxic derivative). Using site-directed mutagenesiswith two synthetic oligonucleotides, a single point mutation, E₂₂₄>A,was introduced into plasmid pLitBoNTA to inactivate the proteolyticactivity responsible for BoNT A neurotoxicity resulting in plasmidpLitBoNTAME224A (Kurazono et al., “Minimal Essential Domains SpecifyingToxicity of the Light Chains of Tetanus Toxin and Botulinum NeurotoxinType A,” J. Biol. Chem. 267:14721-14729 (1992); Lacy et al., “CrystalStructure of Botulinum Neurotoxin Type A and Implications for Toxicity,”Nat. Struct. Biol. 5:898-902 (1998); Agarwal et al., “StructuralAnalysis of Botulinum Neurotoxin Type E Catalytic Domain and Its MutantGlu212>Gln Reveals the Pivotal Role of the Glu212 Carboxylate in theCatalytic Pathway,” Biochemistry 43:6637-6644 (2004), which are herebyincorporated by reference in their entirety). Atoxic derivativesproduced in this way will better preserve the structural moietiesresponsible for toxin immunogenicity, trafficking, and cell recognitionsites.

A third full-length BoNT A derivative was designed to test the utilityof the genetic engineering methodology to produce fusion proteins, usingGFP as an example. The sequence encoding the minimal catalytic domain ofthe BoNT A LC (Kadkhodayan et al., “Cloning, Expression, and One-StepPurification of the Minimal Essential Domain of the Light Chain ofBotulinum Neurotoxin Type A,” Protein Expr. Purif 19:125-130 (2000),which is hereby incorporated by reference in its entirety) was excisedfrom plasmid pLitBoNTA and replaced with a GFP-coding sequence to createplasmid pLitGFPBoNTAHC encoding a GFP derivative of BoNT A (gfpd). TheGFP-encoding sequence was obtained by PCR with two syntheticoligonucleotides on the plasmid pEGFP-N3 (Clontech, Palo Alto, Calif.).The fusion protein was specifically designed to preserve structuralfeatures responsible for cell binding and intracellular trafficking.

All intermediate DNA constructs as well as plasmids pLitBoNTA,pLitBoNTAME224A, and pLitGFPBoNTAHC were checked by multiple restrictiondigests. pLitBoNTA and pLitBoNTAME224A were sequenced with twelve BoNTA-specific synthetic oligonucleotides, while pLitGFPBoNTAHC wassequenced with ten BoNT A-specific synthetic oligonucleotides and twoGFP-specific oligonucleotides as primers, resulting in a set ofoverlapping sequences which covered all coding parts of all of the aboveplasmids. All sequences were demonstrated to be free of unexpectedmutations.

Example 14 Expression of the Recombinant BoNT A Derivatives in E. coli

Expression plasmids were transfected into E. coli Rosetta-gami B (DE3)competent cells (Novagen, Cat. #71136-3) by the heat-shock methodaccording to manufacturer protocol. Bacterial cultures were grown in LBmedia containing 50 mg/l carbenicillin, 15 mg/l kanamycin, 12.5 mg/ltetracycline and 34 mg/l chloramphenicol. Various conditions, affectingthe plasmid copy number per cell without and with addition ofL-arabinose (0.01% final concentration) to the bacterial medium weretested. All bacterial cultures used for protein expression were grown at37° C. until reaching OD@600 nm ˜0.3-0.4. Prior to the induction of theexpression, bacterial cultures were split to test influence of thetemperature on the yield and quality of the expressed product. Uponinduction (OD@600 nm ˜0.5-0.7), cultures were grown at 37° C., 25° C.,and 12° C. Final IPTG concentration in the growth medium used forinduction was 0.5 mM. For the time-course study, samples of the cultureat 1, 3, 6, 9, and 12 hours after induction were collected and analyzed.Under the optimal conditions the E. coli cultures were incubatedovernight in the presence of L-arabinose at 37° C. until reaching OD˜0.4 @ 600 nm. The temperature of the bacterial suspensions was thenlowered to 12° C. over one hour, and IPTG was added to a finalconcentration of 0.5 mM. After induction, culture growth was allowed tocontinue in a shaker incubator at 12° C. for six more hours. Thebacterial pellet was then harvested by centrifugation on Sorwall GS3rotor (7000 rpm, 25 min., 4° C.) and processed for recombinant proteinisolation. Cells kept on ice were resuspended in BugBuster lysis reagent(Novagen, Cat.#70584-4) with the volume ratio cell paste:BugBustersolubilization reagent 1:5. The nucleic acid degradation reagentbenzonaze was used instead of the mixture sonication (Novagen,Cat.#70746-3), 1000 U/ml final concentration, recombinant lysozyme(Novagen, Cat.#71110-4), 50 U/ml final concentration, and a cocktail ofprotease inhibitors “Complete” (Roche (Switzerland), Cat.#1697498), 1tablet/50 ml final concentration were added simultaneously to the pastein the process of resuspension. Approximately 30 minutes afterresuspension, the non-viscous lysate was cleared by centrifugation onSorwall SS34 rotor (17000 rpm, 25 min, 4° C.) and processed for thefurther protein purification.

An E. coli expression system was the first tested for a number ofreasons. First, other laboratories have reported expression ofrecombinant partial length BoNT A domains in this system (Rigoni et al.,“Site-Directed Mutagenesis Identifies Active-Site Residues of the LightChain of Botulinum Neurotoxin Type A,” Biochem. Biophys. Res. Commun.288:1231-1237 (2001); Chaddock et al., “Expression and Purification ofCatalytically Active, Non-Toxic Endopeptidase Derivatives of ClostridiumBotulinum Toxin Type A,” Protein Expr. Purif. 25:219-228 (2002); Lalliet al., “Functional Characterization of Tetanus and BotulinumNeurotoxins Binding Domains,” J. Cell Sci. 112:2715-2724 (1999);Kadkhodayan et al., “Cloning, Expression, and One-Step Purification ofthe Minimal Essential Domain of the Light Chain of Botulinum NeurotoxinType A,” Protein Expr. Purif. 19:125-130 (2000); Lacy et al.,“Recombinant Expression and Purification of the Botulinum NeurotoxinType A Translocation Domain,” Protein Expr. Purif. 11: 195-200 (1997),which are hereby incorporated by reference in their entirety). A secondreason for selecting an E. coli expression system is that manyrecombinant proteins can be expressed in E. coli with good yield andstability. A third reason is that non-canonical E. coli codons in theBoNT A sequence can be overcome by utilizing a bacterial strain carryinga plasmid encoding tRNA for the rare codons. A fourth reason is thattoxicity of the full-length BoNT A to the host can be minimized by usingan expression plasmid that allows regulation of the transition from lowto medium plasmid copy numbers. Fifth, proper disulfide bridge formationin the recombinant proteins can be optimized by utilizing an E. colistrain with trxB⁻ gor⁻ mutations. Sixth, degradation of recombinantproteins in the host can be minimized by utilizing an E. coliRosetta-gami strain with lon⁻ ompT⁻ mutations, in which two majorproteolytic enzymes are inactivated.

Expression plasmids were obtained by single-step subcloning of thecoding portion of BoNT A derivatives—td, ad, and gfpd into theexpression vector pETcoco2 (Novagen, San Diego, Calif.). The resultingconstructs contain DNA, encoding sequence MHHHHHHGAS . . . (SEQ ID NO:44) and flanked with NheI unique restriction site in front of the firstnative methionine codon. The pETcoco system combines the advantages ofT7 promoter-driven protein expression with the ability to controlplasmid copy number. The pETcoco vectors are normally maintained at onecopy per cell. In the single-copy state, pETcoco clones are extremelystable, which is especially important for target genes that are toxic tothe host. Copy number can be amplified to 20-50 copies per cell by theaddition of L-arabinose to the culture medium. The pETcoco vectors inλDE3 lysogenic hosts can be induced to increase expression of the targetgene by as much as 2,500-fold over background when IPTG is added to theculture media. A 6-His tag was added to each recombinant protein toenable affinity purification. The affinity tag was added to theN-terminus, because prior studies found that addition of an affinity tagto the C-terminus results in loss of the toxin's physiological activity(Shapiro et al., “Identification of a Ganglioside Recognition Domain ofTetanus Toxin Using a Novel Ganglioside Photoaffinity Ligand,” J. Biol.Chem. 272:30380-30386 (1997), which is hereby incorporated by referencein its entirety), while adding a hexahistidine tag to the N-terminusallowed expression and purification of the light chain domain withretained enzymatic activity (Kadkhodayan et al., “Cloning, Expression,and One-Step Purification of the Minimal Essential Domain of the LightChain of Botulinum Neurotoxin Type A,” Protein Expr. Purif. 19:125-130(2000), which is hereby incorporated by reference in its entirety).

All expression constructs were transformed into E. coli Rosetta-gami B(DE3) competent cells (Novagen) and were grown in LB media containingampicillin, kanamycin, tetracycline, and chloramphenicol. Ampicillin wasadded to select for colonies carrying pETcoco derived bla marker,kanamycin and tetracyclin were added to select for thioredoxin (trxB)and glutathione reductase (gor) mutations, thus improving the chancesfor proper disulfide bond formation in the E. coli cytoplasm (Derman etal., “Mutations that Allow Disulfide Bond Formation in the Cytoplasm ofEscherichia Coli,” Science 262:1744-1747 (1993); Prinz et al., “The Roleof the Thioredoxin and Glutaredoxin Pathways in Reducing ProteinDisulfide Bonds in the Escherichia Coli Cytoplasm,” J. Biol. Chem.272:15661-15667 (1997), which are hereby incorporated by reference intheir entirety). Chloramphenicol was added to the medium to select forcolonies containing helper plasmids that provide tRNAs for rare codons,thereby increasing the expression of proteins such as BoNT A encoded byDNA with codons non-canonical for E. coli.

Multiple conditions were tested to optimize expression of the BoNT Afull length derivatives. Cultures were grown with and withoutL-arabinose in the media, and different IPTG concentrations wereevaluated for induction. Incubation temperatures and time were alsooptimized for BoNT derivative expression. Under optimal conditions, theE. coli cultures were incubated overnight in the presence of L-arabinoseat 37° C. until reaching OD ˜0.4 at 600 nm. The temperature of thebacterial suspensions was then lowered to 12° C. over one hour, and IPTGwas added to a final concentration 0.5 mM. After induction, culturegrowth was allowed to continue in a shaker incubator at 12° C. for sixmore hours. The bacterial pellet was then harvested by centrifugation,lysed with BugBuster lysis reagent (Novagen) in the presence of nucleicacid degradation reagent benzonaze (Novagen), lysozyme, and a cocktailof protease inhibitors. The lysate was cleared by centrifugation andpurified by incubation with a Ni-NTA affinity resin. The supernatant andeluate from the Ni-NTA agarose were run on 8% SDS PAGE gels, andanalyzed by Western blotting with polyclonal antibodies raised againstthe full-length BoNT A inactivated toxioid. Rosetta-gami B (DE3) E. colitransformed with the empty vector was used as the negative control.Native BoNT A in SDS-PAGE loading buffer was used as the positivecontrol.

FIG. 4 illustrates the results of E. coli expression and purificationprotocols for BoNT A td. The expressed protein was soluble and could bepurified using the chelate affinity tag. However, the molecular weightof the recombinant BoNT A td full length propeptide expressed wassignificantly lower than that of the native full-length BoNT Apropeptide. Extensive proteolysis was observed with all purification andexpression protocols tested in E. coli, even when the toxin derivativeswere expressed by the cells in the single-copy plasmid state. Thisinstability may be related to the systems available in E. coli forpost-translational processing of proteins, with improper folding anddisulfide bonding making the recombinant toxins susceptible todegradation. Similar results were obtained when attempting to expressthe atoxic (ad) and GFP- (gfpd) derivatives of BoNT A in E. coli. Theproblems encountered with E. coli based expression systems with respectto native protein folding and extensive proteolysis of the recombinantproduct, may be resolved by modification and optimization of the E. coliexpression system.

Example 15 Expression of BoNT A Derivatives in Baculovirus-Based System

Bac-to-Bac® baculovirus expression system (Invitrogen, Cat.#10359-016)was used for the generation of the recombinant baculoviruses. A protocolfor the insect cell culture was taken from the manual supplied with thekit. Recombinant donor plasmids were transformed into Max EfficiencyDH10Bac™ competent cells (Invitrogen, Cat.#10361-012). Coloniescontaining recombinant bacmid were identified by disruption of the lacZαgene and selected by the absence of developing blue color, while growingon the plate with the chromogenic substrate Bluo-gal (Invitrogen,Cat.#15519-028). High molecular weight DNA was isolated from theselected colonies on DNA plasmid purification system (Qiagen,Cat.#12245). Transposition of the DNA of interest into baculovirusgenome was confirmed by PCR on high molecular weight DNA witholigonucleotides CBA14 and CBA17, resulting in amplification of 1170b.p. DNA band in samples where transposition took place. Bacmids wereused to transfect serum-free medium adapted Sf9 insect cells(Invitrogen, Cat.#11496-015) to produce baculoviruses. Transfection wasperformed by the following protocol: 9×10⁵ cells were seeded per one35-mm well in 2 ml of unsupplemented Grace's insect cell culture medium(Invitrogen, Cat.#11595-030). Cells from a 3 to 4 day-old suspensionculture in mid-log phase with a viability of >97% were used forexperiment. Cells were attached to the plastic at 27° C. for at leastone hour in advance and transfected with the lipophylic complexes,formed after mixing bacmid with Cellfectin® transfection reagent(Invitrogen, Cat.#10362-010), according to the protocol supplied by themanufacturer. 72 hours after transfection, the supernatant containingrecombinant baculoviruses was harvested and separated from the cells bylow-speed centrifugation (Sorwall GS 3 Rotor, 2000 rpm, 20 min, 4° C.).The supernatant represents the primary baculoviral stock. Amplificationof this baculoviral stock and viral plaque assay was performed accordingto the protocol supplied by the manufacturer. Experiments related toidentification of the optimal MOI and time-course studies of recombinantprotein expression were also performed according to the manufacturerrecommendations.

For the purpose of protein expression, Sf9 cells were grown as a shakenculture in a SF900 II serum-free medium (Invitrogen, Cat.#10902-088) at27° C. in humidified atmosphere. At the density of the cell culture˜1.2×10⁶/ml, baculovirus stock in the same medium was added tosuspension at MOI˜0.1. Incubation continues for another ˜50 hours, afterwhich medium was separated from the cells by centrifugation (Sorwall GS3 Rotor, 2000 rpm, 20 min, 4° C.) and further processed for the proteinpurification by the procedure outlined below. Sf9 cells are verysensitive to growth conditions. They require a constant temperature of27±1° C., good aeration of shaking cultures, and a sterile environment.If ambient temperatures rise above 27° C., refrigeration is required inthe incubator used. An incubator sufficiently large to producesufficient quantities of BoNT derivatives for biological testing isrecommended.

To avoid poisoning of the insect cell host, the BoNT A td construct wasmodified by adding a signal peptide to provide for secretion of therecombinant proteins to the medium. Targeting the recombinant toxins forsecretion also resulted in proper disulfide bond formation between thetoxin's light and heavy chains. Improvements to this expression systemwere tested as described infra.

To increase the total yield of the recombinant protein, donorrecombinant baculovirus plasmids and bacmids were generated with anexpression cassette that allows expression of the recombinant protein tobe driven by two separate and independent promoters simultaneously, p10and PH (donor plasmid pFastBac™ Dual, Invitrogen, Cat.#10712-024).

To stabilize and increase the titer of the recombinant baculoviralstock, an approach outlined in BaculoDirect® Expression System protocol(Invitrogen (Carlsbad, Calif.), Cat.#12562-021) was used that allowsnegative selection to remove non-recombinant baculovirus that tend toappear in amplified stocks over the time. To improve purification of thetoxins, the affinity of the recombinantly expressed proteins for Ni-NTAresin was increased by generating additional BoNT constructs with longerN-terminal His tags.

The advantages of a baculovirus expression system include properdisulfide bridge formation which has been demonstrated for numerousrecombinant proteins in this system; protein purification, which isfacilitated when serum-free culture medium is utilized and the expressedproteins contain a short secretory signal and affinity tag;physiological activity similar to native progenitors can be retained inthe expressed products; and the absence of endotoxins endogenous to E.coli, which facilitates biological testing and therapeutic use of theexpressed proteins (Allen et al., “Recombinant Human Nerve Growth Factorfor Clinical Trials: Protein Expression, Purification, Stability andCharacterisation of Binding to Infusion Pumps,” J. Biochem. Biophys.Methods. 47:239-255 (2001); Curtis et al., “Insect Cell Production of aSecreted form of Human Alpha(1)-Proteinase Inhibitor as a BifunctionalProtein which Inhibits Neutrophil Elastase and has Growth Factor-LikeActivities,” J. Biotechnol. 93:35-44 (2002), which are herebyincorporated by reference in their entirety). The disadvantages of thissystem are its cost, time-consuming procedures, and generally the yieldof proteins is not as high as in E. coli. Furthermore, because theregulated exocytosis machinery is well preserved across eukaryoticspecies from yeast to mammals, expression of BoNT A in this system canpotentially lead to the host poisoning and cellular death. Nonetheless,since Clostridial neurotoxins are known to pass through epithelial cellsby transcytosis without any toxic affects (Simpson, “Identification ofthe Major Steps in Botulinum Toxin Action,” Annu. Rev. Pharmacol.Toxicol. 44:167-193 (2004); Park et al., “Inhalational Poisoning byBotulinum Toxin and Inhalation Vaccination with Its Heavy-ChainComponent,” Infect. Immun. 71:1147-1154 (2003), which are herebyincorporated by reference in their entirety), and the toxin constructsdescribed herein are designed to remain in the single-chain propeptideform until processed to dichain mature form by enterokinase, this systemis worth further testing.

Plasmid constructs for expression of BoNT A derivatives in this systemwere subcloned into the donor vector pFastBac™1 (Invitrogen). Tofacilitate secretion of the recombinant proteins into the media and toallow purification of the recombinant proteins on Ni-NTA agarose, a DNAsequence coding the gp64 signal peptide and a hexahistidine affinity tagMPMLSAIVLYVLLAAAAHSAFAAMVHHHHHHSAS . . . (SEQ ID NO: 45), flanked withunique NheI restriction site in front of the first native methioninecodon, was introduced by cloning of PCR product into all constructs.FIG. 5 provides a schematic representation of the BoNT A derivativestargeted for expression in the baculovirus system. The signal peptideshown in the illustrated recombinant proteins is removed by secretaseprocessing during intracellular trafficking (von Heijne, “Signals forProtein Targeting Into and Across Membranes,” Subcell. Biochem. 22:1-19(1994), which is hereby incorporated by reference in its entirety).Expression of the genes in the vector pFastBac™1 is controlled by theAutographa californica multiple nuclear polyhedrosis virus (AcMNPV)polyhedrin (PH) promoter. Recombinant donor plasmids were transformedinto DH10Bac® E. coli competent cells. Once the pFastBac™ basedexpression plasmid is in cellular cytoplasm, transposition occursbetween the arms of mini-Tn7 element flanking the expression cassette inpFastBac™ based vector and the mini-attTn7 target site on thebaculovirus shuttle vector (bacmid), already present in the cells. Thisevent generates a recombinant bacmid. Transposition requires additionalproteins supplied by helper plasmids also present in competent cells.Selection of the recombinant bacmid clones was performed visually (bysize and color). The molecular nature of the isolated DNAs was confirmedby PCR with the specific oligonucleotide primers.

Recombinant bacmids and negative control bacmids (obtained as a resultof transposition with empty donor plasmids) were transfected into Sf9insect cells with the lipophilic reagent Cellfectin (Invitrogen). After96 hours the recombinant baculoviral stock was harvested and used forinfection of freshly seeded Sf9 cells.

Secondary baculoviral stock was used for multiple purposes, whichinclude, testing the expression of recombinant proteins, amplifyingrecombinant baculoviruses and generating tertiary stock for future use,calculating the titer of recombinant baculoviruses, identifying theoptimal ratio for multiplicity of infection (MOI), and establishing theoptimal time course for protein expression. Baculovirus titer wascalculated for each newly amplified baculoviral stock. For all BoNT Aconstructs tested, the optimal MOI was found to be ˜0.1 pfu per cell andthe optimal time for the protein harvest was found to be ˜50 hours afterinfection. When recombinant proteins were allowed to accumulate in themedia for 72 hours or longer, a significant portion of the recombinantprotein was degraded due to virus-induced cellular lysis.

FIG. 6 illustrates the protein expression results for the toxic (td),atoxic (ad), and GFP-linked (gfpd) full-length propeptide derivatives ofBoNT A (cultures harvested at 50 hrs). All recombinant proteins weresoluble and secreted into the media, could be purified by binding to theaffinity resin, and have the expected mobility on SDS PAGE comparable tothe mobility of single chain wt BoNT A. The recombinant BoNT Aderivatives expressed using these conditions were free of degradationproducts recognized by the polyclonal antibody.

Example 16 Enterokinase Processing and LC-HC Disulfide Bridges

FIG. 7 illustrates a dosage-titration curve for cleavage of thepropeptide constructs with recombinant enterokinase (rEK), using the tdderivative as an example. For the processing of the single-chain (SC)protein, different amounts have been applied to the BoNT A td. Using 0.5U of the enzyme at 20° C. for 8 hours was found to completely digest 1μg of the sc BoNT A td.

To evaluate whether the disulfide bridges between the light and heavychains of the recombinant proteins were properly formed, the recombinantpropeptide derivatives were compared on reducing and non-reducing gelsafter digestion with excess rEK. Western blots were probed withpolyclonal antibodies raised against native full-length BoNT A toxoid.The results of this experiment, shown in FIGS. 8A and 8B, demonstratethat all of the recombinant propeptides were processed into atwo-subunit form by rEK, and that the subunits could be readilyseparated after reduction of the disulfide bridges, as expected.

Expression of a GFP-linked derivative of BoNT A is demonstrated by thegreen fluorescence of Sf9 cells 12 hours after infection with therecombinant baculovirus expressing BoNT A gfpd (FIGS. 8C and 8D). Thesignificant difference in the background of FIG. 8C (recombinantbaculovirus expressing BoNT A gfpd with secretion signal) versus FIG. 8D(recombinant baculovirus expressing GFP control), is believed to resultfrom the secretion of the fluorescent recombinant protein into themedia.

Example 17 Purification of the Recombinant BoNT A Derivatives

Methods to purify reasonable quantities of the full-length BoNT Aderivatives were developed using BoNT A td as an example. Though therecombinant protein was found to bind to Ni-NTA resin (FIG. 5), theaffinity was not sufficient to establish a one step purification scheme.The protein bound to the Ni-NTA resin in 5 mM imidazole, but was elutedfrom the affinity column by 40 mM imidazole. At this concentration ofimidazole, there are other proteins present in the eluate, and thereforeadditional steps are needed to separate recombinant protein fromcontaminants.

Similar results were observed with the minimal essential domain of BoNTA expressed and purified in E. coli (Kadkhodayan et al., “Cloning,Expression, and One-Step Purification of the Minimal Essential Domain ofthe Light Chain of Botulinum Neurotoxin Type A,” Protein Expr. Purif.19:125-130 (2000), which is hereby incorporated by reference in itsentirety). The stable minimal essential domain of the LC expressed withtwo 6-His tags on the N- and C-termini of the protein was eluted fromthe Ni-NTA column by 90 mM imidazole, still a relatively lowconcentration of affinity eluant. Poor accessibility of the affinitytags may explain these difficulties. Interestingly, there were twofractions of the same protein from the affinity column, with the secondfraction eluted in 250 mM imidazole. While 90 mM imidazole fraction wasenzymatically active, as was shown in SNAP-25 peptide cleavage assay,the higher concentration imidazole eluate was not. Denaturation of theprotein may explain its absence of activity and high affinity for thechelate matrix.

A multi-step protocol was developed for purifying BoNT A td tohomogeneity. Sf9 cells (viable cells count before infection ˜1.2·10⁶/ml)grown at 27° C. in SF900II serum-free media in humidified atmosphere at125 rpm in shaking culture were harvested and separated from the medium.At ˜50 hours after infection with recombinant baculovirus (MOI˜0.1), themedium was collected, precipitated with ammonium sulfate orconcentrated, dialyzed, and subjected to sequential DEAE-sepharosechromatography, MonoS chromatography, Ni-NTA affinity chromatography,and FPLC-based gel filtration chromatography. FIG. 9 illustrates theresults of protein purification. The yield of the pure recombinantprotein was 0.35 mg from one liter of serum-free medium. The pureprotein eluted from the final gel-filtration column was competent forfurther processing with rEK. After the rEK cleavage, chloride ionscontaining buffer need to be substituted with phosphate or HEPES-basedbuffer to avoid instability of the recombinant toxin derivatives.

Approximately 2 ml of supernatant or cleared lysate was concentrated onAmicon Ultra-4 centrifugal filter device (Millipore, Cat.#UFC803024) to˜1 ml. The concentration procedure was done in parallel with multiplerounds of buffer substitution aimed at removing substances which couldcontribute to Ni²⁺-stripping from the affinity resin. Final buffercomposition was equal to the Ni-NTA Equilibration Buffer (infra). 20 μlof Ni-NTA suspension equilibrated in the Ni-NTA Equilibration Buffer(1:1 v/v) was added to the sample, followed by the sample incubation onthe rotating platform for 1 hour. After incubation, affinity matrix wasseparated from the supernatant by centrifugation (3000 g, 1 min), andwashed three times with Ni-NTA Equilibration Buffer, followed bycentrifugation. The washing buffer was aspirated and the resin wasresuspended in ˜200 μl of SDS-PAGE loading buffer. The liquid was usedfor the further analysis by SDS PAGE and Western blotting.

TABLE 1 BoNT A td Purification Composition of the buffers used: DEAESepharose Equilibration Buffer: 20 mM NaH₂PO₄, 1 mM EDTA, pH 8.0 DEAESepharose Wash Buffer: 50 mM NaCl, 20 mM NaH₂PO₄, pH 8.0 DEAE SepharoseElution Buffer: 500 mM NaCl, 20 mM NaH₂PO₄, pH 8.0 Mono S EquilibrationBuffer: 20 mM NaH₂PO₄, pH 6.8 Mono S Wash Buffer: 25 mM NaCl, 20 mMNaH₂PO₄, pH 6.8 Mono S Elution Buffer: 300 mM NaCl, 20 mM NaH₂PO₄, pH6.8 Ni-NTA Equilibration Buffer: 5 mM imidazole, 50 mM NaH₂PO₄, 300 mMNaCl, pH 8.0 Ni-NTA Wash Buffer I: Same as above but made up with 10 mMimidazole Ni-NTA Wash Buffer II: Same as above but made up with 20 mMimidazole Ni-NTA Elution Buffer: Same as above but made up with 60 mMimidazole HiLoad 16/60 Superdex 200PG Equilibration Buffer: 50 mM NaCl,20 mM Tris-HCl, pH 7.5

All concentration, dialysis, and chromatography steps were performed at4° C. 300 ml of the conditioned insect medium was either concentrated onthe stirred ultrafiltration cell (Millipore, Cat.#5123) with Ultracel100-KDa cutoff membrane (Millipore, Cat.#14432) to the final volume 5ml, or the total protein from the medium was precipitated by addition ofammonium sulfate (60 g/100 ml) with slow stirring. Pellet was separatedfrom the supernatant by centrifugation (5000 g, 20 min, 4° C.) anddissolved in 5 ml of DEAE-Sepharose Equilibration Buffer. Recombinantprotein recovered from the first procedure was less denatured, and thisprocedure is preferable for future work. Scale-up production of the BoNTderivatives for biological testing could be accomplished with TangentialFlow Concentration System (Pellicon 2, Millipore, Cat.#XX42PLK60) whichwould enable large volumes of the media to be processed. During membraneconcentration or ammonium sulfate precipitation, an insolubleprecipitate forms from the pluronic surfactant included in the SF900 IImedia (to prevent cellular aggregation and to reduce shearing forces,thereby improving the stability of the Sf9 insect cells). The insolublepluronic pellet was removed by centrifugation of theconcentrate/ammonium sulfate precipitate (5000 g, 20 min, 4° C.), andrecombinant toxin in the pellet was recovered by extracting twice withDEAE-Sepharose Equilibration Buffer, followed by centrifugation.

Recovered combined supernatant was dialyzed against 100× volumes ofDEAE-Sepharose Equilibration Buffer for 16 hours, separated from theresidual pellet by centrifugation, and loaded on a column (1.5×10 cm)packed with DEAE-Sepharose Fast Flow (Amersham Biosciences,Cat.#17-0709-01) pre-equilibrated in the same buffer at a buffer flowrate of 0.5 ml/min. The column was washed with ˜15 volumes of DEAESepharose Wash Buffer and then a linear gradient of 100 ml DEAESepharose Wash Buffer:100 ml DEAE Sepharose Elution Buffer was applied.4-ml fractions were collected and their content was analyzed by PAGE andWestern blotting. Fractions containing recombinant protein were combinedand dialyzed against 100× volumes of the Mono S Equilibration Buffer for16 hours. Resulting combined dialyzate was cleared by centrifugation andloaded at 1 ml/min on MonoS 5/50 GL FPLC column (Amersham Biosciences,Cat.#17-5168-01), pre-equilibrated in the same buffer. Column was washedwith 100 ml of Mono S Wash Buffer and the linear gradient of 100 ml MonoS Wash Buffer:100 ml Mono S Elution Buffer was applied. 2-ml fractionswere collected and their content was analyzed by PAGE and Westernblotting.

The fractions containing recombinant protein were combined, concentratedon the stirred ultrafiltration cell with Ultracel 100-KDa cutoffmembrane to the final volume of 20 ml with sequential buffer change toNi-NTA Equilibration Buffer. Combined fractions were loaded on a 1×4 cmcolumn with Ni-NTA affinity resin (Novagen, Cat.#70666) pre-equilibratedin the same buffer at a buffer flow rate of 1 ml/min. The column wassequentially washed with 100 ml of Ni-NTA Wash Buffer I, followed by 100ml of Ni-NTA Wash Buffer II, and protein was eluted from the column by50 ml of Ni-NTA Elution Buffer. All fractions were analyzed by PAGE andWestern blotting. Elution fractions enriched in recombinant protein wereconcentrated sequentially on the stirred ultrafiltration cell withUltracel 100-KDa cutoff membrane followed by Amicon Ultra-4 centrifugalfilter device (Millipore, Cat.#UFC803024) to a final volume of 1 ml andloaded on the FPLC HiLoad 16/60 Superdex 200PG gel filtration column(Amersham Biosciences, Cat. #17-1069-01), equilibrated with HiLoad 16/60Superdex 200PG Equilibration Buffer. The buffer flow rate was 1 ml/minand 1-ml fractions from the column were collected and analyzed by PAGEand Western blotting.

The multi-step protocol developed for BoNT A td purification provided ayield ˜0.35 mg of pure protein per liter of serum-free medium. It isbelieved that this procedure can be optimized to provide yields in therange of 0.7-0.9 mg/l. Several reasons may explain the relatively lowyield in the purification of BoNT A td: 1) Significant amounts of therecombinant toxin may be lost due to non-specific adsorption to thebrand-new separation media; 2) The delays which occurred betweenpurification steps may have resulted in degradation of recombinanttoxins. These delays were impossible to avoid during the initialpurification attempts, because it was necessary to analyze therecombinant products before proceeding to the next purification step.The following modifications, aimed at simplifying and improving thecurrent purification scheme, were tested.

Example 18 Biological Testing of the Recombinant BoNT A Derivatives

Two types of experiments were performed to assess whether therecombinant toxins retained the biological activities of native toxin.These were performed using the BoNT A td derivative, which was producedin sufficient quantities for biological testing. In the first test,recombinant BoNT A td was administered to mice by the intravenous route(˜1 ng per mouse) and the time-to-death was monitored. Death wasobserved approximately 12 minutes after injection. Prior symptoms ofmuscular weakness or paralysis were not obvious. In the second test,recombinant BoNT A td was added to mouse phrenic nerve-hemidiaphragmpreparations, and its ability to inhibit acetylcholine release evoked bystimulation of the nerve trunk (0.2 Hz) was evaluated by monitoringmuscle twitch. At a concentration of ˜1×10⁻¹¹ M, recombinant BONT A tdproduced neuromuscular blockade in 167±17 min (n=4). To insure that theblockade could be attributed to a botulinum toxin-type action, a finalexperiment was done, in which the polypeptide was pre-incubated (roomtemperature, 60 min) with rabbit antiserum raised against the carboxyterminal half of the native BoNT A heavy chain (i.e. receptor-bindingdomain). In these experiments (n=3), there was no neuromuscularblockade, even when the tissues were monitored for ca. 400 minutes. Thepharmaceutical preparation marketed by Allergan Inc., as “BoTox”produces neuromuscular blockade in 100 minutes at a concentration ofapproximately 1×10⁻¹¹ M (60 Units per ml). The BoNT A, B, and Grecombinant products produced by Rummel (supra) require 60 to 1000 timesmore BoNT to effect neuromuscular blockade in a similar timeframe.

Example 19 Preparation and Modification of the BoNT Gene Constructs

DNA and protein sequences for Clostridial toxins are accessed from theGene bank. Constructs encoding full-length toxins are available from anumber of laboratories. These known sequences and constructs provide anefficient starting point for the planned genetic manipulations.

The first type of mutation introduced is designed to improve toxinstability by site-directed mutations of low specificityprotease-sensitive residues within the light-heavy chain junctionregion, thereby reducing susceptibility to non-specific activation andpoisoning of the host organism. The second type of mutation will beintroduced to create a highly specific enterokinase cleavage sitebetween the light and heavy chains, thereby enabling external control ofthe cleavage event leading to toxin maturation. The third type ofmutation to be introduced is designed to silently inactivate DNAelements affecting RNA transcription and protein expression in thesystem of choice. The fourth type of modification is designed tointroduce unique restriction sites that enable easy manipulation of thetoxin gene, its protein products, and chimeric proteins which may becreated as required.

The modified BoNT A constructs used to produce the BoNT A toxicderivative (td) described infra demonstrates the feasibility of thesemethods. The objective is to determine how to best adapt the methodsdeveloped for BoNT A to producing other Clostridial neurotoxins, and inthe process optimize the methodology and create a library of toxinderivatives with customized biological properties. Molecular cloningtechniques are generally known in the art, and other full-lengthneurotoxins have successfully been cloned (Ichtchenko et al.,“Alpha-Latrotoxin Action Probed with Recombinant Toxin: ReceptorsRecruit Alpha-Latrotoxin but do not Transduce an Exocytotic Signal,”EMBO J. 17:6188-6199 (1998), which is hereby incorporated by referencein its entirety).

Example 20 Creation and Expression of Recombinant BoNT MoleculesMinimally Modified to Elimante Toxicity

To create atoxic derivatives (“ad”) that most closely resemble thenative toxin with respect to their structure and physiologic activity, asingle amino acid point mutation is introduced into the active site ofthe toxin's metalloprotease catalytic domain. Though most toxin featuresin this molecule remain the same as in the native toxin, it is devoid oftoxicity, because it is unable to cleave its substrate in the synapticexocytosis machinery. The atoxic derivatives thus created are superiorto other BoNT preparations being developed as vaccines, because of theirstructural similarity to native toxin, and their ability to generate animmune response at diverse sites along the native toxin's absorption andtrafficking route. Because these derivatives are likely to compete withnative toxin for the same binding sites and trafficking pathways, theymay also be superior to antibody preparations as antidotes to BoNTpoisoning.

The cloning and expression strategies developed can be duplicated asclosely as possible in applying the methods to BoNT B and E, therebyminimizing the possibility of creating significant molecular alterationsin the atoxic derivatives which might decrease their therapeuticpotential. The validity of this assumption is demonstrated supra withthe BoNT A atoxic derivative (ad), which has been shown to beessentially identical to native BoNT A with respect to expression level,antibody recognition, disulfide bonding, cleavage with enterokinase, andbinding to Ni-NTA affinity resin.

An outline of the steps necessary to produce atoxic derivatives of BoNTB and E is as follows. Constructs encoding the atoxic derivatives (ad)of BoNT B and E are obtained by site-directed mutagenesis of BoNT B andBoNT E td constructs, using procedures established for BoNT A ad, asdetailed supra. Expression constructs for BoNT B ad and BoNT E ad in thedifferent expression systems to be tested are prepared using a protocolsimilar to that established for BoNT A ad, as detailed supra. Theexpression system, purification protocol, and rEK-cleavage protocol forBoNT B and E ad replicate the optimized procedure developed for BoNT Atd and ad, as outlined supra. The expression and purification systemchosen to produce the atoxic derivatives is based on the quality andyield produced by the expression systems tested.

The atoxic derivatives are tested in a substrate cleavage assay usingSNAP 25 or VAMP as substrates. Though no residual proteolytic activityin the single-amino acid mutated atoxic derivatives is expected, if therate of substrate hydrolysis for any particular atoxic derivative issignificantly higher than zero, a second amino acid residue,corresponding to His₂₂₇ in BoNT A, is mutated at the toxin's active sitebefore proceeding for its further biological and functionalcharacterization.

Prophetic Example 21 Preparation of DNA Starting Material for BoNTSerotypes B and E

DNA template for all BoNT serotypes for PCR amplification can beobtained from either pure Clostridium cultures (serotype-specific) orsoil-derived anaerobic cultures from which mixed genomic DNA as astarting material may be prepared. High fidelity Platinum®Pfx polymeraseis used for all PCR reactions to minimize amplification errors. BoNT Band E serotypes are described in subsequent examples.

Prophetic Example 22 Constructs for BoNT B and E

A set of oligonucleotides similar to those used for obtaining thefull-length coding sequence of BoNT A td may be designed for BoNT B andE, using sequences available from Gene bank (accession number M81186 forBoNT B and X62683 for BoNT E). Sequences are carefully evaluated forunwanted DNA regulatory elements and other features that could affectprotein expression in E. coli, baculovirus, and Pichia pastorisexpression systems, and such elements eliminated by silent site-directedmutagenesis. Additional mutations targeted to remove low-specificityproteolysis sites in the toxin's LC-HC junction are introduced, and tointroduce an enterokinase cleavage site in the LC-HC junction region.Based on the toxin sequence alignments and domain structure illustratedin FIGS. 1-3, gene regions which can be modified without affecting therecombinant toxin's biological properties are identified, and Nhe I,XbaI, KpnI, and XhoI restriction sites are introduced, similar to thedesign scheme executed for BoNT A td. If such mutations are impossibleto make through silent mutagenesis, restriction sites are introduced vianeutral amino acid insertion into structurally flexible portions of theprotein sequence. Any redundant restriction sites created are eliminatedby silent site-directed mutagenesis. BoNT DNA sequences that can causepremature termination of gene transcription in the expression systems orinterfere with the protein expression are also modified. Expression inPichia pastoris (Henikoff et al., “Sequences Responsible forTranscription Termination on a Gene Segment in SaccharomycesCerevisiae,” Mol. Cell Biol. 4:1515-1520 (1984); Imiger et al.,“Different Classes of Polyadenylation Sites in the Yeast SaccharomycesCerevisiae,” Mol. Cell Biol. 11:3060-3069 (1991); Scorer et al., “TheIntracellular Production and Secretion of HIV-1 Envelope Protein in theMethylotrophic Yeast Pichia Pastoris,” Gene 136:111-119 (1993), whichare hereby incorporated by reference in their entirety) requires theelimination of such sequences by designing a set of PCR oligonucleotideprimers which can suppress premature termination of transcription fromAT-rich templates. These procedures produce constructs containingmodified coding sequences for BoNT B and BoNT E td, which are used insubsequent expression studies.

Endonuclease restriction digests are used to check all intermediate DNAproducts. The final full-length DNA is sequenced to prove absence ofunwanted mutations.

Molecular biocomputing software, supplied by the DNAstar is used toanalyze and compare DNA and protein sequences. This will also optimizethe creation of synthetic oligonucleotides and optimal reactionconditions for all reactions of PCR amplification.

Prophetic Example 23 Expression, Purification, and Biochemical Analysisof Toxic Derivatives

Expression and purification of full-length, functionally active toxinshas proven difficult in laboratories using alternative construct designsand expression systems. The ideal construct and expression systempreferably do not segregate Clostridial toxins, because they containcoding sequences non-typical for the host; are not poisoned by entry ofactive toxin into the cytosol where it may disrupt the apparatus forregulated exocytosis, which is similar in most eukaryotes; and allownormal post-translational modification of the expressed toxins,particularly formation of disulfide bridges.

Two expression systems were tested for each toxin serotype A:baculovirus and E. coli. Because the baculovirus expression system wasfound to be most effective for expressing full-length BoNT A td, thiswas used as a starting point and benchmark for all the toxins. Thoughmuch concentration was centered on the baculovirus expression system,alternatives were evaluated, taking scale-up and cost intoconsideration, and work can be performed to optimize expression of allserotypes in these systems, as well as in other expression systems suchas Pichi pastoris.

Work that was performed to optimize expression and purification aredescribed supra. The effect of these modifications on nativity of thetoxin was evaluated in each case.

Prophetic Example 24 E. coli Expression System

Attempts to produce full-length BoNT A in E. coli resulted in a majorC-terminally truncated propeptide which was significantly smaller thanexpected for the BoNT A propeptide (FIG. 4). In the future, theC-terminal composition of this product will be analyzed bymicrosequencing, identifying putative proteolytic cleavage sitesspecific to the E. coli system, and redesigning the pETcoco expressionconstruct with amino acid substitutions designed to suppress thiseffect. It is possible the proteolysis site is similar to thatrecognized by trypsin, which has been demonstrated to cleave within theC-terminal BoNT A receptor-binding domain when applied in excessiveamounts (Chaddock et al., “Expression and Purification of CatalyticallyActive, Non-Toxic Endopeptidase Derivatives of Clostridium BotulinumToxin Type A,” Protein Expr. Purif. 25:219-228 (2002), which is herebyincorporated by reference in its entirety). Expression of re-designedconstruct will use the advanced Rosetta-gami B (DE3) E. coli strain, asdescribed infra.

Prophetic Example 25 Targeting Secretion to the Periplasm

Targeting recombinant proteins for secretion to the E. coli periplasmcan improve stability and post-translational disulfide bond formation.The coding portion of the BoNT A td sequence will be subcloned intopET39b(+) vector (Novagen, Cat.#70090-3) which contains the signalrequired for export and periplasmic folding of target proteins. Thissystem is designed for cloning and expression of peptide sequences fusedwith the 208 amino acids DsbA-Tag™. DsbA is a periplasmic enzyme thatcatalyzes the formation and isomerization of disulfide bonds (Rietsch etal., “An In vivo Pathway for Disulfide Bond Isomerization in Escherichiacoli,” Proc. Natl. Acad. Sci. USA 93:13048-13053 (1996); Sone et al.,“Differential In vivo Roles Played by DsbA and DsbC in the Formation ofProtein Disulfide Bonds,”J. Biol. Chem. 272:10349-10352 (1997);Missiakas et al., “The Escherichia coli DsbC (xprA) Gene Encodes aPeriplasmic Protein Involved in Disulfide Bond Formation,” EMBO J.13:2013-2020 (1994); Zapun et al., “Structural and FunctionalCharacterization of DsbC, a Protein Involved in Disulfide Bond Formationin Escherichia coli,” Biochemistry 34:5075-5089 (1995); Raina et al.,“Making and Breaking Disulfide Bonds,” Annu. Rev. Microbiol. 51:179-202(1997), which are hereby incorporated by reference in their entirety).It is possible that the degradation of BoNT A described infra occurs asa result of E. coli incompetence to properly form disulfide bridges forproteins which accumulate in the cytoplasm. The DsbA vector may enhancesolubility and proper folding of recombinant BoNTs in the non-reducingperiplasmic environment (Collins-Racie et al., “Production ofRecombinant Bovine Enterokinase Catalytic Subunit in Escherichia coliUsing the Novel Secretory Fusion Partner DsbA,” Biotechnology 13:982-987(1995), which is hereby incorporated by reference in its entirety).Though the yield of recombinant proteins targeted to the periplasm isusually low, periplasmic expression in E. coli is worth continuedconsideration.

Prophetic Example 26 Pichia pastoris Expression System

Multi-copy Pichia pastoris expression kit (Invitrogen, Cat.#K1750-01) isused to obtain recombinant proteins. Recombinant plasmid on the backboneof the vector pPIC9K, carrying gene of interest and targeted forincorporation into Pichia genome is digested by restriction endonucleaseSal I for linearization and transformed in the Pichia strains GS115 andKM71 by spheroplasting method with zymolyase, according to the suppliedmanufacturer's protocol. Primary and secondary rounds of thetransformants selection on the histidine-deficient medium and in thepresence of Geneticin is performed according to the same protocol.Protein expression is induced by the addition of methanol (0.5% finalconcentration) into the culture medium. Disrupted cells and medium areanalyzed by SDS-PAGE and Western blotting.

Though the baculovirus expression system was found to providesatisfactory level of protein expression of BoNT A, recombinant proteinexpression in Pichia pastoris (methylotrophic yeast capable ofmetabolizing methanol as its sole carbon source) was evaluated becauseof the multiple reports describing successful expression in this system,including fragments of botulinum neurotoxin type A (Byrne et al.,“Purification, Potency, and Efficacy of the botulinum Neurotoxin Type ABinding Domain from Pichia pastoris as a Recombinant Vaccine Candidate,”Infect. Immun. 66:4817-4822 (1998), which is hereby incorporated byreference in its entirety). This system has the advantages of low cost,post-translational modification of the recombinant proteins typical foreukaryotes, and low amounts of naturally secreted products, whichfacilitate purification of the recombinant proteins. The Pichiaexpression system also can provide better yields than the baculovirussystem. Disadvantages of the Pichia system include cumbersome proceduresof cloning into the Pichia genome and selection of multiple-copyrecombinants, the possibility of extensive glycosylation of somerecombinant proteins, and the possibility of premature termination ofRNA transcripts synthesized from AT-rich templates, a knowncharacteristic of the Clostridial toxin genes. These unwanted internalDNA features in BoNT genes were eliminated at the cloning stage. Thesystem was the first to be tested with BoNT A td to establish benchmarksfor comparison to other expression systems.

Prophetic Example 27 Engineering of the Expression Constructs Targetedfor Secretion

The coding part of the modified N-terminally 6-His tagged BoNT A td wassubcloned into vector pPIC9K, which provides the alpha-factor secretionsignal from S. cerevisiae in the expression plasmid. This should resultin secretion of the expressed protein into the medium. The construct waslinearized by restriction endonuclease Sal I and transfected byspheroplasting method into KM71 and GS115 strains of Pichia pastoris.Primary selection of the transformants was performed by testing abilityof the cells to grow on histidine-deficient media, trait deficient inthe wild-type cells. Second round of selection was performed in thepresence of antibiotic Geneticin which allowed the identification ofclones with multiple inserts of the gene of interest by the correlationbetween the number of the copies of the gene of interest and increasedconcentration of Geneticin in the growth medium. The ability ofidentified clones to express protein of interest will be tested bygrowing cells in the methanol-containing medium.

Prophetic Example 28 Lengthening of the His Affinity Tag

The length of the histidine affinity tag at the N-termini of the BoNT Atd were increased, and two more constructs—8-His and 12-His tagged weretested for their ability to confer higher affinity for Ni-NTA agarose inthe recombinant BoNT products. This approach has been used successfullyfor other recombinant proteins which showed a similar decreased affinityfor Ni-NTA affinity purification media (Ichtchenko et al.,“Alpha-Latrotoxin Action Probed with Recombinant Toxin: ReceptorsRecruit Alpha-Latrotoxin but do not Transduce an Exocytotic Signal,”EMBO J. 17:6188-6199 (1998); Rudenko et al., “Structure of the LDLReceptor Extracellular Domain at Endosomal pH,” Science 298:2353-2358(2002), which are hereby incorporated by reference in their entirety).If improved purification of recombinant BoNT A td can be achieved, atleast two steps of ion-exchange chromatography can be omitted from thecurrent purification scheme.

Example 29 Engineering the Non-Expression Plasmid pLitBoNTAME224AContaining the Full-Length Sequence of BoNT A ad

The plasmid encoding full-length BoNT A ad cDNA withprotease-inactivating mutation E224>A was created by the site-directedmutagenesis of the plasmid pLitBoNTA with phosphorylatedoligonucleotides

CBA18: (SEQ ID NO: 46) 5′-pCCCGCGGTGACATTAGCACATGCACTTATACATGCTGG andCBA19: (SEQ ID NO: 47) 5′-pCATGTGCTAATGTCACCGCGGGATCTGTAGCAAATTTGusing GeneTailor™ Site-Directed Mutagenesis System (Invitrogen,Cat.#12397-014) and Platinum® Pfx DNA Polymerase (Invitrogen,Cat.#11708-021), according to the protocol supplied by the manufacturer.The size of pLitBoNTAME224A is 6712 b.p. with 3900 b.p. coding sequence.

Example 30 Engineering of the Non-Expression Plasmid pLitGFPBoNTAHC,Containing Full-Length Sequence of BoNT A gfpd

The plasmid pLitGFPBoNTAHC, encoding chimeric protein where minimalessential catalytic domain of the BoNT A light chain was substitutedwith the GFP was created by the following protocol: 742 b.p. PCRproduct, obtained on plasmid pEGFP-N3 (Clontech, Cat.#632313) witholigonucleotides

CBA20: (SEQ ID NO: 48) 5′-ATTAAGGATCCTGTGAGCAAGGGCGAGGAGCTGTTCACCG andCBA21: (SEQ ID NO: 49)5′-TATGAATTCAAACAATCCAGTAAAATTTTTCTTGTACAGCTCGTCCA TGCCand digested with restriction endocucleases BamHI and EcoRI andsubcloned into pre-digested and dephosphorylated vector pLitBoNTALC,resulting in plasmid pLitGFPLC. 2615 b.p. DNA fragment from the vectorpLitBoNTAHC, digested with restriction endonucleases XbaI and ApaI wassubcloned into pre-digested and dephosphorylated vector pLitGFPLC,resulting in plasmid pLitGFPBoNTAHC. The size of pLitGFPBoNTAHC is 6216b.p. with 3404 b.p. of coding sequence.

Example 31 Engineering of the Expression Plasmids pETCBoNTAME224A andpETCGFPBoNTAHC for the Expression of BoNT A ad and BoNT A gfpd in E.coli

The plasmids were obtained by subcloning DNA fragments isolated frompLitmus-based vectors digested with NheI and NotI into pre-digested anddephosphorylated expression vector pETcoco2 (Novagen, Cat.#71148-3) andresulted in 16,194 b.p. BoNT A E224>A mutant expression vectorpETCBoNTAME224A and 15,699 b.p. BoNT A chimeric vector pETCGFPBoNTAHC,where minimal essential catalytic domain of the BoNT A light chain wassubstituted with the GFP.

Example 32 Engineering of the Donor Plasmids pFBSecBoNTAME224A andpFBSecGFPBoNTAHC for the Expression of BoNT A ad and BoNT A gfpd inBaculovirus Expression System

The plasmids were obtained by the following protocol: 112 b.p. PCRproduct synthesized on plasmid pBac-3 (Novagen, Cat.#70088-3) witholigonucleotides

CBA 22: (SEQ ID NO: 50) 5′-TAAGCGCGCAGAATTCTCTAGAATGCCCATGTTAAGCGCTATTGand CBA23: (SEQ ID NO: 51) 5′-TAAGCTAGCGTGATGGTGGTGATGATGGACCATGGCCand digested with restriction endonucleases BssHII and NheI wassubcloned into pre-digested and dephosphorylated pLitmus-based vectors,resulting in plasmids pLitSecBoNTAME224A and pLitSecGFPBoNTAHC. DNAfragments from these vectors, obtained as a result of the digest withBssHII and NotI, were subcloned into pre-digested and dephosphorylatedvector pFastBaC™1 (Invitrogen, Cat.#10360-014), resulting in 8764 b.p.pFBSecBoNTAME224A and 8568 b.p. pFBSecGFPBoNTAHC donor plasmids.

Example 33 Chimera Proteins that Target the Cytosol ofNeurotoxin-Affected Neurons

A genetic engineering platform designed to produce BoNT antidotes thatcan effectively target the cytoplasm of BoNT-affected neurons has beendeveloped. Antidotes designed pursuant to this platform have thepotential to be effectively administered to a subject for extended timeperiods after exposure to toxic Clostridial neurotoxin, and wouldimprove the practical logistics of administering antidote to largepopulations in an emergency setting. Using genetic constructs of theisolated BoNT A light chain, protein motifs are introduced to bind,inactivate, or otherwise mark toxic wild-type Clostridial neurotoxin(e.g., Clostridium botulinum) light chain for elimination from thecytosol of neurotoxin-affected neurons. Chimeric light chains withoptimized antidote activity can subsequently be recombined withderivitized constructs of the Clostridial neurotoxin heavy chain toproduce full-length Clostridial neurotoxin chimeras that can deliverantidote activity to the cytosol of Clostridial neurotoxin-affectedneurons. Expression systems can be developed and tested (as describedabove) to ensure that the structural features and post-translationalmodifications responsible for native Clostridial neurotoxin traffickingare preserved. Produced Clostridial neurotoxin antidotes of this sortcan effectively target neurotoxin-affected neurons when administered byoral or inhalational routes and can be used to rescue patients alreadyexperiencing symptoms of Clostridial neurotoxin intoxication (e.g.,patients on an artificial respirator).

Using the plasmid encoding atoxic BoNT A light chain, a first library ofBoNT A ad light chain chimeras containing SNARE motif peptidessubstituting light chain alpha-helix regions has been designed andcreated. The SNARE motif is recognized by all seven BoNT serotypes, andprior work has demonstrated physiological exocytosis (Rossetto et al.,“SNARE Motif and Neurotoxins,” Nature 372:415-416 (1994), which ishereby incorporated by reference in its entirety). The chimeras areconstructed to retain the interface responsible for BoNT light chaindimerization (Segelke et al., “Crystal Structure of Closridium BotulinumNeurotoxin Protease in a Product-Bound State: Evidence for NoncanonicalZinc Protease Activity,” Proc. Natl. Acad. Sci. USA 101:6888-6893(2004), which is hereby incorporated by reference in its entirety),allowing them to preferentially bind to wild-type light chains andpotentially inactivate or otherwise destabilize the toxic neurotoxin.Engineering of non-expression plasmids containing the full-lengthsequence of BoNT A (atoxic derivative, ad) with non-native SNARE motifpeptides (illustrated in FIG. 11) to produce the light chain chimericlibraries is described in the following paragraphs.

Light Chain of BoNT A

The plasmid encoding mutated light chain of BoNT A cDNA withmetalloprotease-inactivating mutation E224>A (pLitBoNTALCME224A) wascreated by the site-directed mutagenesis of the plasmid pLitBoNTALC withphosphorylated oligonucleotides

CBA18: (SEQ ID NO: 46) 5′-pCCCGCGGTGACATTAGCACATGCACTTATACATGCTGG andCBA19: (SEQ ID NO: 47) 5′-pCATGTGCTAATGTCACCGCGGGATCTGTAGCAAATTTGusing GeneTailor™ Site-Directed Mutagenesis System (Invitrogen, Cat.#12397-014) and Platinum® Pfx DNA Polymerase (Invitrogen, Cat.#11708-021), according to the protocol supplied by the manufacturer. Theresulting plasmid pLitBoNTALCME224A is 4042 b.p. with a 1230 b.p. codingsequence.

Chimera 1

The non-expression plasmid pLitBoNTACH1, containing the full-lengthsequence of BoNT A ad with three SNARE motif peptides substituting BoNTA light chain alpha-helix 1, was created by site-directed mutagenesis ofthe plasmid pLitBoNTAME224A with phosphorylated oligonucleotides

CBCH1: (SEQ ID NO: 52)5′-pGAGTTGTTCGCCTTGCTCATCCAACATCTGCAACGCGTCAGCTCGGTCATCCAACTCTGTACTTAAATATGTTGAATCATAATATGAAACTGG and CBCH2: (SEQ ID NO:53) 5′-pGAGCGCGAAATGGATGAAAACCTAGAGCAGGTGAGCGGCCGAGGAATACCATTTTGGGGTGGAAGTACAATAGATACAGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit (Stratagene,Cat.#200502) with modification. The ExSite™ DNA polymerase blendincluded in the kit was substituted with a blend consisting of 75% ofTaKaRa LA Taq DNA polymerase (Takara, Cat.#RR002A) and 25% of Platinum®Pfx polymerase (Invitrogen, Cat.#11708-021). The mutagenesis reactionand selection of the mutant plasmid were performed according to theprotocol, included in the original Exsite™ PCR-Based Site-DirectedMutagenesis Kit. For selection purposes, two de novo endonucleaserestriction sites-MluI and XhoI-were introduced into the plasmid.

Chimera 2

The non-expression plasmid pLitBoNTACH2, containing the full-lengthsequence of BoNT A ad with two SNARE motif peptides substituting BoNT Alight chain alpha-helix 4, was created by site-directed mutagenesis ofthe plasmid pLitBoNTAME224A with phosphorylated oligonucleotides

CBCH3: (SEQ ID NO: 54)5′-pCGCGTCTGCCCTATCGTCTAGTTCATCTATAAACTTTGCATCATGT CCCCC and CBCH4: (SEQID NO: 55) 5′-pTTACAAATGCTAGACGAACAGGGAGAGCAGCTCGAGAGGCTTAATA AAGCTAAATCAATAGTAGGTACTACTGCusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications, described above.

Chimera 3

The non-expression plasmid pLitBoNTACH3, containing the full-lengthsequence of BoNT A ad with five SNARE motifs peptides substituting BoNTA light chain alpha-helices 1 and 4, was created by site-directedmutagenesis of the plasmid pLitBoNTACH1 with phosphorylatedoligonucleotides

CBCH3: (SEQ ID NO: 54)5′-pCGCGTCTGCCCTATCGTCTAGTTCATCTATAAACTTTGCATCATGT CC CCC and CBCH4:(SEQ ID NO: 55) 5′-pTTACAAATGCTAGACGAACAGGGAGAGCAGCTCGAGAGGC TTAATAAAGCTAAATCAATAGTAGGTACTACTGCusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above.

Chimera 4

The non-expression plasmid pLitBoNTACH4, containing the full-lengthsequence of BoNT A ad with three SNARE motif peptides substituting lightchain alpha-helices 4 and 5, was created by site-directed mutagenesis ofthe plasmid pLitBoNTACH2 with phosphorylated oligonucleotides

CBCH5: (SEQ ID NO: 56)5′-pGCTTACTTGTTCCAAATTCTCGTCCATCTCTGAATGCAGTAGTACC TAC TATTGATTTAGC andCBCH6: (SEQ ID NO: 57) 5′-pGGCCGTCTCCTATCTGAAGATACATCTGGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site Eco52I was introduced into the plasmid.

Chimera 5

The non-expression plasmid pLitBoNTACH5, containing the full-lengthsequence of BoNT A ad with six SNARE motif peptides substituting BoNT Alight chain alpha-helices 1, 4, and 5, was created by site-directedmutagenesis of the plasmid pLitBoNTA CH2 with phosphorylatedoligonucleotides

CBCH5: (SEQ ID NO: 56)5′-pGCTTACTTGTTCCAAATTCTCGTCCATCTCTGAAGCAGTAGTACCT AC TATTGATTTAGC andCBCH6: (SEQ ID NO: 57) 5′-pGGCCGTCTCCTATCTGAAGATACATCTGGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site Eco52I was introduced into the plasmid.

Chimera 6

The non-expression plasmid pLitBoNTACH6, containing the full-lengthsequence of BoNT A ad with four SNARE motif peptides substituting BoNT Alight chain alpha-helices 4, 5, and 6, was created by site-directedmutagenesis of the plasmid pLitBoNTACH4 with phosphorylatedoligonucleotides

CBCH7: (SEQ ID NO: 58) 5′-pAATTCATCCATGAAATCTACCGAAAATTTTCC and CBCH8:(SEQ ID NO: 59) 5′-pCTTTGAACAGGTGGAGGAATTAACAGAGATTTACACAGAGGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site EcoRI was introduced into the plasmid.

Chimera 7

The non-expression plasmid pLitBoNTACH7, containing the full-lengthsequence of BoNT A ad with five SNARE motif peptides substituting BoNT Alight chain alpha-helices 4, 5, 6, and 7, was created by site-directedmutagenesis of the plasmid pLitBoNTA CH6 with phosphorylatedoligonucleotides

CBCH9: (SEQ ID NO: 60) 5′-pTCGAGCTCTGTGTAAATCTCTGTTAATTCC and CBCH10:(SEQ ID NO: 61) 5′-pGGACATGCTGGAGAGTGGGAATCTTAACAGAAAAACATATTTGAAT TTTGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site XhoI was introduced into the plasmid.

Chimera 8

The non-expression plasmid pLitBoNTACH8, containing the full-lengthsequence of BoNT A ad with seven SNARE motif peptides substituting BoNTA light chain alpha-helices 1, 4, 5, and 6, was created by site-directedmutagenesis of the plasmid pLitBoNTACH5 with phosphorylatedoligonucleotides

CBCH7: (SEQ ID NO: 58) 5′-pAATTCATCCATGAAATCTACCGAAAATTTTCC and CBCH8:(SEQ ID NO: 59) 5′-pCTTTGAACAGGTGGAGGAATTAACAGAGATTTACACAGAGGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site EcoRI was introduced into the plasmid.

Chimera 9

The non-expression plasmid pLitBoNTACH9, containing the full-lengthsequence of BoNT A ad with eight SNARE motif peptides substituting BoNTA light chain alpha-helices 1, 4, 5, 6, and 7, was created bysite-directed mutagenesis of the plasmid pLitBoNTACH8 withphosphorylated oligonucleotides

CBCH9: (SEQ ID NO: 60) 5′-pTCGAGCTCTGTGTAAATCTCTGTTAATTCC and CBCH10:(SEQ ID NO: 61) 5′-pGGACATGCTGGAGAGTGGGAATCTTAACAGAAAAACATATTTGAAT TTTGusing Exsite™ PCR-Based Site-Directed Mutagenesis Kit with themodifications described above. For the selection purposes, de novoendonuclease restriction site XhoI was introduced into the plasmid.

Green Fluorescence Protein

The non-expression plasmid pLitEGFP, containing the full-length sequenceof GFP for the fusions with BoNT A light chain, BoNT A light chain ad,and BoNT A light chain ad chimeric derivatives, was created bysubcloning ˜750 b.p. product obtained by PCR on plasmid pEGFP-N3(Clontech, Cat.#6080-1) with oligonucleotides

EGFPs: (SEQ ID NO: 62)5′-TATTACGCGTGCGCGCTATGAATTCTATAAGTTGCTAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGG and EGFPa: (SEQ ID NO: 63)5′-ATTAGGGCCCCTATTACTTGTACAGCTCGTCCATGCCGAGAGTGATC CCand digested with restriction endonucleases MluI and ApaI into vectorpLitmus38i (NEB, Cat.#N3538S) pre-digested with Mul and ApaI anddephosphorylated. The size of the resulting pLitEGFP was 3479 b.p.

Light Chain of BoNT A td Fused with EGFP

The non-expression vector pLitBoNTALCEGFP carrying light chain of BoNT Atd, fused with EGFP, was created by subcloning the 1296 b.p. DNAfragment obtained from the digest of the plasmid pLitBoNTALC withrestriction endonucleases BssHII and EcoRI into vector pLitEGFPpre-digested with BssHII and EcoRI and dephosphorylated. The size of theresulting plasmid was ˜4800 b.p.

Light Chain of BoNT A ad Fused with EGFP

The non-expression vector pLitBoNTAME224ALCEGFP carrying the sequence ofthe light chain of BoNT A ad, fused with EGFP, was created by subcloningthe 1296 b.p. DNA fragment obtained from the digest of the plasmidpLitBoNTAME224A with restriction endonucleases BssHII and EcoRI intovector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated.The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 1 Fused with EGFP

The non-expression vector pLitBoNTACHIEGFP, carrying the sequence of theBoNT A ad light chain with three SNARE motif peptides substituting BoNTA light chain alpha-helix 1, fused with EGFP, was created by subcloningthe 1296 b.p. DNA fragment obtained from the digest of the plasmidpLitBoNTACH1, with restriction endonucleases BssHII and EcoRI intovector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated.The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 2 Fused with EGFP

The non-expression vector pLitBoNTACH2EGFP, carrying the sequence of theBoNT A ad light chain with two SNARE motif peptides substituting BoNT Alight chain alpha-helix 4, fused with EGFP, was created by subcloningthe 1296 b.p. DNA fragment obtained from the digest of the plasmidpLitBoNTA CH2 with restriction endonucleases BssHII and EcoRI intovector pLitEGFP pre-digested with BssHII and EcoRI and dephosphorylated.The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 3 Fused with EGFP

The non-expression vector pLitBoNTACH3EGFP, carrying the sequence of theBoNT A ad light chain with five SNARE motif peptides substituting BoNT Alight chain alpha-helices 1 and 4, fused with EGFP, was created bysubcloning the 1296 b.p. DNA fragment obtained from the digest of theplasmid pLitBoNTACH3 with restriction endonucleases BssHII and EcoRIinto vector pLitEGFP pre-digested with BssHII and EcoRI anddephosphorylated. The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 4 Fused with EGFP

The non-expression vector pLitBoNTACH4EGFP, carrying the sequence of theBoNT A ad light chain with three SNARE motif peptides substituting BoNTA light chain alpha-helices 4 and 5, fused with EGFP, was created bysubcloning the 1296 b.p. DNA fragment obtained from the digest of theplasmid pLitBoNTACH4 with restriction endonucleases BssHII and EcoRIinto vector pLitEGFP pre-digested with BssHII and EcoRI anddephosphorylated. The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 5 Fused with EGFP

The non-expression vector pLitBoNTA CH5EGFP, carrying the sequence ofthe BoNT A ad light chain with six SNARE motif peptides substitutingBoNT A light chain alpha-helices 1, 4, and 5, fused with EGFP, wascreated by subcloning the 1296 b.p. DNA fragment, obtained from thedigest of the plasmid pLitBoNTACH5 with restriction endonucleases BssHIIand EcoRI into vector pLitEGFP pre-digested with BssHII and EcoRI anddephosphorylated. The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimer 6 Fused with EGFP

The non-expression vector pLitBoNTA CH6EGFP, carrying the sequence ofthe BoNT A ad light chain with four SNARE motif peptides substitutingBoNT A light chain alpha-helices 4, 5, and 6, fused with EGFP, wascreated by subcloning 1296 b.p. DNA fragment, obtained from theincomplete digest with EcoRI of the 2019 b.p. DNA fragment, obtainedfrom the plasmid pLitBoNTACH6, digested with restriction endonucleasesBssHII and AlwNI, into vector pLitEGFP pre-digested with BssHII andEcoRI and dephosphorylated. The size of the resulting plasmid was ˜4800b.p.

Light Chain of Chimera 7 Fused with EGFP

The non-expression vector pLitBoNTACH7EGFP, carrying the sequence of theBoNT A ad light chain with five SNARE motif peptides substituting BoNT Alight chain alpha-helices 4, 5, 6, and 7, fused with EGFP, was createdby subcloning the 1296 b.p. DNA fragment, obtained from the incompletedigest with EcoRI of the 2019 b.p. DNA fragment, obtained from theplasmid pLitBoNTACH7 digested with restriction endonucleases BssHII andAlwNI into vector pLitEGFP pre-digested with BssHII and EcoRI anddephosphorylated. The size of the resulting plasmid was ˜4800 b.p.

Light Chain of Chimera 8 Fused with EGFP

The non-expression vector pLitBoNTA CH8EGFP, carrying the sequence ofthe BoNT A ad light chain with seven SNARE motif peptides substitutingBoNT A light chain alpha-helices 1, 4, 5, and 6, fused with EGFP, wascreated by subcloning the 1296 b.p. DNA fragment obtained from theincomplete digest with EcoRI of the 1754 b.p. DNA fragment obtained fromthe plasmid pLitBoNTACH8 digested with restriction endonucleases BssHIIand HincII into vector pLitEGFP pre-digested with restrictionendonucleases BssHII and EcoRI and dephosphorylated. The size of theresulting plasmid was ˜4800 b.p.

Light Chain of Chimera 9 Fused with EGFP

The non-expression vector pLitBoNTACH9EGFP, carrying the sequence of theBoNT A ad light chain with eight SNARE motif peptides substituting BoNTA light chain alpha-helices 1, 4, 5, 6, and 7, fused with EGFP, wascreated by subcloning the 1296 b.p. DNA fragment obtained from theincomplete digest with EcoRI of the 1754 b.p. DNA fragment obtained fromthe plasmid pLitBoNTACH9 digested with restriction endonucleases BssHIIand HincII, into vector pLitEGFP pre-digested with restrictionendonucleases BssHII and EcoRI and dephosphorylated. The size of theresulting plasmid was ˜4800 b.p.

Sindbis Expression Vector—EGFP

The Sindbis expression vector pSinEGFP, carrying the EGFP sequence wascreated by subcloning the ˜750 b.p. DNA fragment obtained from theplasmid pLitEGFP sequentially digested with restriction endonucleaseEcoRI filled-in with Klenow fragment, and digested with restrictionendonuclease ApaI into vector pSinRep5 (Invitrogen, Cat.#K750-1)pre-digested with restriction endonucleases StuI and ApaI anddephosphorylated. The size of the resulting plasmid was ˜10,250 b.p.

Sindbis Expression Vector—BoNT A td Light Chain Fused with EGFP

The Sindbis expression vector pSinBoNTALCEGFP, carrying the sequence ofBoNT A td light, chain fused with EGFP, was created by subcloning the˜2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTALCEGFP,digested with restriction endonucleases NheI and ApaI into vectorpSinRep5 pre-digested with restriction endonucleases XbaI and ApaI anddephosphorylated. The size of the resulting plasmid was ˜11,550 b.p.

Sindbis Expression Vector—BoNT A ad Light Chain Fused with EGFP

The Sindbis expression vector pSinBoNTAME224ALCEGFP, carrying thesequence of BoNT A ad light, chain fused with EGFP was created bysubcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTAME224ALCEGFP digested with restriction endonucleases NheI andApaI into vector pSinRep5 pre-digested with restriction endonucleasesXbaI and ApaI and dephosphorylated. The size of the resulting plasmidwas ˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 1 Fused with EGFP

The Sindbis expression vector pSinBoNTACH1EGFP, carrying the sequence ofBoNT A ad light chain with three SNARE motif peptides substituting BoNTA light chain alpha-helix 1, fused with EGFP, was created by subcloningthe ˜2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH1EGFPdigested with restriction endonucleases NheI and ApaI into vectorpSinRep5 pre-digested with restriction endonucleases XbaI and ApaI anddephosphorylated. The size of the resulting plasmid was ˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 2 Fused with EGFP

The Sindbis expression vector pSinBoNTACH2EGFP, carrying the sequence ofBoNT A ad light chain with two SNARE motif peptides substituting BoNT Alight chain alpha-helix 4, fused with EGFP, was created by subcloningthe ˜2,050 b.p. DNA fragment obtained from the plasmid pLitBoNTACH2EGFP,digested with restriction endonucleases NbeI and ApaI into vectorpSinRep5 (Invitrogen, Cat.#K750-1) pre-digested with restrictionendonucleases XbaI and ApaI and dephosphorylated. The size of theresulting plasmid was ˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 3 Fused with EGFP

The Sindbis expression vector pSinBoNTACH3EGFP, carrying the sequence ofBoNT A ad light chain with five SNARE motif peptides substituting BoNT Alight chain alpha-helices 1 and 4, fused with EGFP, was created bysubcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH3EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 4 Fused with EGFP

The Sindbis expression vector pSinBoNTACH4EGFP, carrying the sequence ofBoNT A ad light chain with three SNARE motif peptides substituting BoNTA light chain alpha-helices 4 and 5, fused with EGFP, was created bysubcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH4EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 5 Fused with EGFP

The Sindbis expression vector pSinBoNTACH5EGFP, carrying the sequence ofBoNT A ad light chain with six SNARE motif peptides substituting BoNT Alight chain alpha-helices 1, 4, and 5, fused with EGFP, was created bysubcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH5EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 6 Fused with EGFP

The Sindbis expression vector pSinBoNTACH6EGFP, carrying the sequence ofBoNT A ad light chain with four SNARE motif peptides substituting BoNT Alight chain alpha-helices 4, 5, and 6, fused with EGFP, was created bysubcloning ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH6EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 7 Fused with EGFP

The Sindbis expression vector pSinBoNTACH7EGFP, carrying the sequence ofBoNT A ad light chain with five SNARE motif peptides substituting BoNT Alight chain alpha-helices 4, 5, 6, and 7, fused with EGFP, was createdby subcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH7EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 8 Fused with EGFP

The Sindbis expression vector pSinBoNTACH8EGFP, carrying the sequence ofBoNT A ad light chain with seven SNARE motif peptides substituting BoNTA light chain alpha-helices 1, 4, 5, and 6, fused with EGFP, was createdby subcloning the ˜2,050 b.p. DNA fragment obtained from the plasmidpLitBoNTACH8EGFP digested with restriction endonucleases NheI and ApaIinto vector pSinRep5 pre-digested with restriction endonucleases XbaIand ApaI and dephosphorylated. The size of the resulting plasmid was˜11,550 b.p.

Sindbis Expression Vector—Light Chain of Chimera 9 Fused with EGFP

The Sindbis expression vector pSinBoNTACH9EGFP, carrying the sequence ofBoNT A ad light chain with eight SNARE motif peptides substituting BoNTA light chain alpha-helices 1, 4, 5, 6, and 7, fused with EGFP, wascreated by subcloning the ˜2,050 b.p. DNA fragment obtained from theplasmid pLitBoNTACH9EGFP digested with restriction endonucleases NheIand ApaI into vector pSinRep5 pre-digested with restrictionendonucleases XbaI and ApaI and dephosphorylated. The size of theresulting plasmid was ˜11,550 b.p.

The Sindbis expression vectors were prepared for RNA synthesis. Theplasmids pSinEGFP, pSinBoNTALCEGFP, pSinBoNTAME224ALCEGFP,pSinBoNTACH1EGFP, pSinBoNTACH2EGFP, pSinBoNTACH3EGFP, pSinBoNTACH4EGFP,pSinBoNTACH5EGFP, pSinBoNTACH6EGFP, pSinBoNTACH7EGFP, pSinBoNTACH8EGFP,and pSinBoNTACH9EGFP were linearized by the digest with restrictionendonuclease NotI. The liniarized plasmids were used for the RNAsynthesis according to the protocol supplied with Sindbis expressionsystem kit (Invitrogen, Cat.#K750-1).

DSGXXS Motif Library

To further mark the antagonist wild-type BoNT A light chain complex forelimination, a second library of light chain chimeras will be produced.This library will incorporate the DSGXXS (SEQ ID NO: 64) motif into thechimeras produced in the first library. The motif DSGXXS is present in avariety of cytosolic proteins and has been shown to target them fordegradation via the proteosome pathway upon its phosphorylation (Cardozoet al., “The SCF Ubiquitin Ligase: Insights Into a Molecular Machine,”Nat. Rev. Mol. Cell Biol. 5:739-751 (2004); Busino et al., “Degradationof Cdc25A by Beta-TrCP During S Phase and In Response to DNA Damage,”Nature 426:87-91 (2003), which are hereby incorporated by reference intheir entirety). The motif will be positioned to cause minimalinterference with the 3D structure of the ancestral protein (i.e.,wild-type BoNT A light chain).

Although the invention has been described in detail for the purposes ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

1. A Clostridium botulinum neurotoxin propeptide comprising: a lightchain region; a heavy chain region, wherein the light and heavy chainregions are linked by a disulfide bond; an intermediate regionconnecting the light and heavy chain regions and comprising a highlyspecific protease cleavage site, wherein said highly specific proteasecleavage site has three or more specific adjacent amino acid residuesthat are recognized by the highly specific protease in order to enablecleavage; a signal peptide coupled to the light chain region, whereinthe signal peptide is suitable to permit secretion of the neurotoxinpropeptide from a eukaryotic cell to a medium; and an affinity taglocated between the signal peptide and the light chain region, whereinthe affinity tag has a sequence of SEQ ID NO:
 45. 2. The propeptideaccording to claim 1, wherein the Clostridium botulinum has a serotypeselected from the group consisting of Clostridium botulinum serotype A,Clostridium botulinum serotype B, Clostridium botulinum serotype C,Clostridium botulinum serotype D, Clostridium botulinum serotype L,Clostridium botulinum serotype F, and Clostridium botulinum serotype G.3. The propeptide according to claim 1, wherein the highly specificprotease cleavage site is an enterokinase cleavage site.
 4. Thepropeptide according to claim 1, wherein the propeptide has nolow-specificity protease cleavage sites in the intermediate region, saidlow-specificity protease cleavage sites having two or less adjacentamino acid residues that are recognized by a protease in order to permitcleavage.
 5. The propeptide according to claim 1, wherein the light andheavy chain regions are not truncated.
 6. The propeptide according toclaim 1, wherein the propeptide has a disabling mutation in an activemetalloprotease site of the propeptide.
 7. The propeptide according toclaim 1, wherein the signal peptide region is a gp64 signal peptide. 8.The propeptide according to claim 2, wherein the Clostridium botulinumis selected from the group consisting of serotype A, serotype L, andserotype F.
 9. The propeptide according to claim 8, wherein the heavychain has no trypsin-susceptible recognition sequences.